PSMA binding ligand-linker conjugates and methods for using

ABSTRACT

Described herein are prostate specific membrane antigen (PSMA) binding conjugates that are useful for delivering therapeutic, diagnostic and imaging agents. Also described herein are pharmaceutical composition containing them and methods of using the conjugates and compositions. Also described are processes for manufacture of the conjugates and the compositions containing them.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is continuation application of U.S. application Ser.No. 15/606,913, filed May 26, 2017, which is a continuation applicationof U.S. application Ser. No. 14/794,482 filed Jul. 8, 2015, which is acontinuation application of U.S. application Ser. No. 12/673,931 filedFeb. 17, 2010, which is a U.S. national counterpart application ofinternational application serial no. PCT/US2008/073375 filed Aug. 15,2008, which claims priority to U.S. Provisional Patent Application Ser.No. 60/956,489 filed on Aug. 17, 2007, and U.S. Provisional PatentApplication Ser. No. 61/074,358 filed on Jun. 20, 2008, the entiredisclosures of which are hereby incorporated by reference.

TECHNICAL FIELD

The invention described herein pertains to compounds and methods fortreating diseases of the prostate, such as prostate cancer and relateddiseases. More specifically, embodiments of the invention describedherein pertain to conjugates of biologically active agents conjugated toPSMA binding ligands.

BACKGROUND

The prostate is one of the male reproductive organs found in the pelvisbelow the urinary bladder. It functions to produce and store seminalfluid which provides nutrients and fluids that are vital for thesurvival of sperm introduced into the vagina during reproduction. Likemany other tissues, the prostate glands are also prone to develop eithermalignant (cancerous) or benign (non-cancerous) tumors. The AmericanCancer Society predicted that over 230,000 men would be diagnosed withprostrate cancer and over 30,000 men would die from the disease in year2005. In fact, prostate cancer is one of the most common male cancers inwestern societies, and is the second leading form of malignancy amongAmerican men. Current treatment methods for prostrate cancer includehormonal therapy, radiation therapy, surgery, chemotherapy, photodynamictherapy, and combination therapy. The selection of a treatment generallyvaries depending on the stage of the cancer. However, many of thesetreatments affect the quality of life of the patient, especially thosemen who are diagnosed with prostrate cancer over age 50. For example,the use of hormonal drugs is often accompanied by side effects such asosteoporosis and liver damage. Such side effects might be mitigated bythe use of treatments that are more selective or specific to the tissuebeing responsible for the disease state, and avoid non-target tissueslike the bones or the liver. As described herein, prostate specificmembrane antigen (PSMA) represents a target for such selective orspecific treatments.

PSMA is named largely due to its higher level of expression on prostatecancer cells; however, its particular function on prostate cancer cellsremains unresolved. PSMA is over-expressed in the malignant prostatetissues when compared to other organs in the human body such as kidney,proximal small intestine, and salivary glands. Though PSMA is expressedin brain, that expression is minimal, and most ligands of PSMA are polarand are not capable of penetrating the blood brain barrier. PSMA is atype II cell surface membrane-bound glycoprotein with ˜110 kD molecularweight, including an intracellular segment (amino acids 1-18), atransmembrane domain (amino acids 19-43), and an extensive extracellulardomain (amino acids 44-750). While the functions of the intracellularsegment and the transmembrane domains are currently believed to beinsignificant, the extracellular domain is involved in several distinctactivities. PSMA plays a role in central nervous system, where itmetabolizes N-acetyl-aspartyl glutamate (NAAG) into glutamic andN-acetyl aspartic acid. Accordingly, it is also sometimes referred to asan N-acetyl alpha linked acidic dipeptidase (NAALADase). PSMA is alsosometimes referred to as a folate hydrolase I (FOLH I) or glutamatecarboxypeptidase (GCP II) due to its role in the proximal smallintestine where it removes γ-linked glutamate from poly-γ-glutamatedfolate and α-linked glutamate from peptides and small molecules.

PSMA also shares similarities with human transferrin receptor (TfR),because both PSMA and TfR are type II glycoproteins. More specifically,PSMA shows 54% and 60% homology to TfR1 and TfR2, respectively. However,though TfR exists only in dimeric form due to the formation ofinter-strand sulfhydryl linkages, PSMA can exist in either dimeric ormonomeric form.

Unlike many other membrane-bound proteins, PSMA undergoes rapidinternalization into the cell in a similar fashion to cell surface boundreceptors like vitamin receptors. PSMA is internalized throughclathrin-coated pits and subsequently can either recycle to the cellsurface or go to lysosomes. It has been suggested that the dimer andmonomer form of PSMA are inter-convertible, though direct evidence ofthe interconversion is being debated. Even so, only the dimer of PSMApossesses enzymatic activity, and the monomer does not.

Though the activity of the PSMA on the cell surface of the prostatecells remains under investigation, it has been recognized by theinventors herein that PSMA represents a viable target for the selectiveand/or specific delivery of biologically active agents, includingdiagnostic agents, imaging agents, and therapeutic agents to suchprostate cells.

SUMMARY OF THE INVENTION

It has been discovered that biologically active compounds that areconjugated to ligands capable of binding to prostate specific membraneantigen (PSMA) via a linker may be useful in the imaging, diagnosis,and/or treatment of prostate cancer, and related diseases that involvepathogenic cell populations expressing or over-expressing PSMA. PSMA isa cell surface protein that is internalized in a process analogous toendocytosis observed with cell surface receptors, such as vitaminreceptors. Accordingly, it has been discovered that certain conjugatesthat include a linker having a predetermined length, and/or apredetermined diameter, and/or preselected functional groups along itslength may be used to treat, image, and/or diagnose such diseases.

In one illustrative embodiment of the invention, conjugates having theformulaB-L-Dare described wherein B is a prostate specific membrane antigen (PSMA)binding or targeting ligand, L is a linker, and D is a drug. As usedherein, the term drug D collectively includes therapeutic agents,cytotoxic agents, imaging agents, diagnostic agents, and the like,unless otherwise indicated or by the context. For example, in oneillustrative configuration, the conjugate described herein is used toeliminate a pathogenic population of cells and therefore the drug D is atherapeutic agent, a cytotoxic agent, and the like. In anotherillustrative configuration, the conjugate described herein is used toimage and/or diagnose a disease or disease state, and therefore the drugD is an imaging agent, a diagnostic agent, and the like. Otherconfigurations are also contemplated and described herein. It is to beunderstood that analogs and derivatives of each of the foregoing B, L,and D are also contemplated and described herein, and that when usedherein, the terms B, L, and D collectively refer to such analogs andderivatives.

In one illustrative embodiment, the linker L may be a releasable ornon-releasable linker. In one aspect, the linker L is at least about 7atoms in length. In one variation, the linker L is at least about 10atoms in length. In one variation, the linker L is at least about 14atoms in length. In another variation, the linker L is between about 7and about 31, between about 7 and about 24, or between about 7 and about20 atoms in length. In another variation, the linker L is between about14 and about 31, between about 14 and about 24, or between about 14 andabout 20 atoms in length.

In an alternative aspect, the linker L is at least about 10 angstroms(Å) in length. In one variation, the linker L is at least about 15 Å inlength. In another variation, the linker L is at least about 20 Å inlength. In another variation, the linker L is in the range from about 10Å to about 30 Å in length.

In an alternative aspect, at least a portion of the length of the linkerL is about 5 Å in diameter or less at the end connected to the bindingligand B. In one variation, at least a portion of the length of thelinker L is about 4 Å or less, or about 3 Å or less in diameter at theend connected to the binding ligand B. It is appreciated that theillustrative embodiments that include a diameter requirement of about 5Å or less, about 4 Å or less, or about 3 Å or less may include thatrequirement for a predetermined length of the linker, thereby defining acylindrical-like portion of the linker. Illustratively, in anothervariation, the linker includes a cylindrical portion at the endconnected to the binding ligand that is at least about 7 Å in length andabout 5 Å or less, about 4 Å or less, or about 3 Å or less in diameter.

In another embodiment, the linker L includes one or more hydrophiliclinkers capable of interacting with one or more residues of PSMA,including amino acids that have hydrophobic side chains, such as Ser,Thr, Cys, Arg, Orn, Lys, Asp, Glu, Gln, and like residues. In anotherembodiment, the linker L includes one or more hydrophobic linkerscapable of interacting with one or more residues of PSMA, includingamino acids that have hydrophilic side chains, such as Val. Leu, Ile,Phe, Tyr, Met, and like residues. It is to be understood that theforegoing embodiments and aspects may be included in the linker L eitheralone or in combination with each other. For example, linkers L that areat least about 7 atoms in length and about 5 Å, about 4 Å or less, orabout 3 Å or less in diameter or less are contemplated and describedherein, and also include one or more hydrophilic linkers capable ofinteracting with one or more residues of PSMA, including Val, Leu, Ile,Phe, Tyr, Met, and like residues are contemplated and described herein.

In another embodiment, one end of the linker is not branched andcomprises a chain of carbon, oxygen, nitrogen, and sulfur atoms. In oneembodiment, the linear chain of carbon, oxygen, nitrogen, and sulfuratoms is at least 5 atoms in length. In one variation, the linear chainis at least 7 atoms, or at least 10 atoms in length. In anotherembodiment, the chain of carbon, oxygen, nitrogen, and sulfur atoms arenot substituted. In one variation, a portion of the chain of carbon,oxygen, nitrogen, and sulfur atoms is cyclized with a divalent fragment.For example, a linker (L) comprising the dipeptide Phe-Phe may include apiperazin-1,4-diyl structure by cyclizing two nitrogens with an ethylenefragment, or substituted variation thereof.

In another embodiment, pharmaceutical compositions are described herein,where the pharmaceutical composition includes the conjugates describedherein in amounts effective to treat diseases and disease states,diagnose diseases or disease states, and/or image tissues and/or cellsthat are associated with pathogenic populations of cells expressing orover expressing PSMA. Illustratively, the pharmaceutical compositionsalso include one or more carriers, diluents, and/or excipients.

In another embodiment, methods for treating diseases and disease states,diagnosing diseases or disease states, and/or imaging tissues and/orcells that are associated with pathogenic populations of cellsexpressing or over expressing PSMA are described herein. Such methodsinclude the step of administering the conjugates described herein,and/or pharmaceutical compositions containing the conjugates describedherein, in amounts effective to treat diseases and disease states,diagnose diseases or disease states, and/or image tissues and/or cellsthat are associated with pathogenic populations of cells expressing orover expressing PSMA.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A. Cell bound radioactivity versus concentration of SK28-^(99m)Tc(K_(d)=18.12 nM) in the presence (▴) or absence (▪) of excess PMPA.

FIG. 1B. In Vitro Binding Studies Using LNCaP Cells and SK33 (14 atomlinker). LNCaP cells containing increasing concentrations ofDUPA-^(99m)Tc in the presence (▴) or absence (▪) of excess PMPA

FIG. 2. Cell bound radioactivity verses concentration of SK28-^(99m)Tc;at 4° C. (▪) and at 37° C. (▴).

FIG. 3A. Plot of cell bound radioactivity versus concentration ofDUPA-Linker-^(99m)Tc imaging agents: (▪) 0-atom linker (K_(d)=171 nM);(▴) 7-atom linker (K_(d)=68 nM); (▾) 14-atom linker (K_(d)=15 nM); (♦)16-atom linker (K_(d)=40 nM).

FIG. 3B. K_(D) values for DUPA-Linker-^(99m)Tc compounds binding toLNCaP cells.

FIG. 4. Plot of days post injection verses tumor volume for LNCaPtumors: (a) 2.5 million+Matrigel; (b) 2.5 million+HC Matrigel; (c) 5million+Matrigel; (d) 5 million+HC Matrigel.

FIG. 5A. Plot of days post injection verses tumor volume for LNCaPtumors (27 mice) and (b) KB cells (5 mice) and A549 cells (5 mice).

FIG. 5B. Plot of days post injection versus tumor volume for KB cells (5mice) and A549 cells (5 mice).

FIG. 6A. Mice (Set 1) previously injected with LNCaP tumors, treatedwith 1 ng/kg SK28-^(99m)Tc (14-atom linker), the left hand image showswhite light images and image shows an overlay of the radioimage with thewhite light image. In each panel, the right mouse was treated with 50mg/kg PMPA (to block PSMA binding) and the left mouse was treatedwithout added PMPA.

FIG. 6B. Mice (Set 2) previously injected with LNCaP tumors, treatedwith 1 ng/kg SK28-^(99m)Tc (14-atom linker), the left hand image showswhite light images and image shows an overlay of the radioimage with thewhite light image. In each panel, the right mouse was treated with 50mg/kg PMPA (to block PSMA binding) and the left mouse was treatedwithout added PMPA.

FIG. 6C. Mice (Set 3) previously injected with LNCaP tumors, treatedwith 1 ng/kg SK28-^(99m)Tc (14-atom linker), the left hand image showswhite light images and image shows an overlay of the radioimage with thewhite light image. In each panel, the right mouse was treated with 50mg/kg PMPA (to block PSMA binding) and the left mouse was treatedwithout added PMPA.

FIG. 6D. Shows a single mouse study for LNCaP tumors imaged using Kodakimager 4 hours after subcutaneous (administered through intraperitoneal)injection of 1 ng/kg SK28-^(99m)Tc showing in the left hand image anoverlay of radioimage with kidney shield and white light image with noshield and in the right hand image an overlay of radioimage with kidneyshield and X-ray image with no shield.

FIG. 7A. Mice previously injected with LNCaP tumors treated usingSK60-^(99m)Tc (zero atom linker). The left image shows white lightimages, the center image shows overlay of radioimage with white lightimage, and the right image shows overlay of radioimage with white lightimage by shielding the kidney of mice.

FIG. 7B. Mice previously injected with KB cells treated usingSK28-^(99m)Tc (14 atom linker). The left image shows white light images,the center image shows overlay of radioimage with white light image, andthe right image shows overlay of radioimage with white light image byshielding the kidney of mice.

FIG. 7C. Mice previously injected with A549 cells treated usingSK28-^(99m)Tc (14 atom linker). The left image shows white light images,the center image shows overlay of radioimage with white light image, andthe right image shows overlay of radioimage with white light image byshielding the kidney of mice.

FIG. 7D. Whole body images of solid tumor xenografts in nu/nu mice taken4 h after injection of 150 μCi DUPA-^(99m)Tc. Overlay of whole-bodyradioimages on white light images of mice bearing LNCaP tumors that weretreated with DUPA-^(99m)Tc in the absence (a, c) or presence (b, d) of100-fold molar excess PMPA. Overlay of radioimages on white light imagesof mice bearing an A549 tumor (e) or a KB tumor (f) that were similarlytreated with DUPA-^(99m)Tc.

FIG. 8A. Bio-distribution data as measured for direct cpm count versestissue for SK28-^(99m)Tc with or without PMPA (as a competitor) onLNCaP, (a) without, (b) with; (c) A549; and (d) KB tumors implanted onaxial of male nude mice.

FIG. 8B. Bio-distribution data just for tumor and kidney forSK28-^(99m)Tc with (b) or without (a) PMPA (as a competitor) on LNCaPtumors implant on axial of male nude mice.

FIG. 8C. Biodistribution studies of DUPA-^(99m)Tc in nu/nu mice bearingLNCaP, A549, or KB tumors.

FIG. 9A. Acute MTD (single dose) showing percentage weight change aftera single dose of SK71; saline alone, 1.1 μmol/kg, 2.3 μmol/kg, 4.5μmol/kg, and 9 μmol/kg.

FIG. 9B. Chronic MTD showing percentage weight change after 5 dosesgiven on alternate days (M, W, F, M, W); saline alone, 2 μmol/kg and 4μmol/kg.

FIG. 10A. Efficacy study showing tumor volume in animals treated withthe conjugate SK71 administered in 5 doses on alternate days (M, W, F,M, W) at 1 μmol/kg.

FIG. 10B. Efficacy study (control group) showing tumor volume in animalstreated with saline alone administered in 5 doses on alternate days (M,W, F, M, W).

FIG. 10C. Efficacy study (competition) showing tumor volume in animalstreated with excess PSMA and the conjugate SK71 administered in 5 doseson alternate days (M, W, F, M, W) at 1 μmol/kg.

FIG. 11. Efficacy Study (1 micromole/kg every other day for 10 days;i.e. 5 doses).

FIG. 12A. [³H]-Thymidine incorporation of LNCaP cells after treatmentwith SK71 (IC₅₀˜2 nM).

FIG. 12B. [³H]-Thymidine incorporation of LNCaP cells after treatmentwith SK77 (IC₅₀˜3 nM).

FIG. 12C. [³H]-Thymidine incorporation of LNCaP cells after treatmentwith SK37 (IC₅₀˜33 nM).

FIG. 12D. [³H]-Thymidine incorporation of LNCaP cells after treatmentwith SK45 (IC₅₀˜29 nM).

FIG. 13A. The effect of treatment with SK71 (1.5 μmol/kg) on tumorvolume in nu/nu mice previously treated with LNCaP cells in HC Matrigel.Treated mice (▪), untreated mice (●), treated mice pre-injected with100-fold molar excess of PMPA (▴).

FIG. 13B. The effect of treatment with SK71 (1.5 μmol/kg) on percentageweight change in nu/nu mice previously treated with LNCaP cells in HCMatrigel. Treated mice (▪), untreated mice (●), treated micepre-injected with 100-fold molar excess of PMPA (▴).

FIG. 13C. The effect of treatment with SK71 (2.0 μmol/kg) on tumorvolume in nu/nu mice previously treated with LNCaP cells in HC Matrigel.Treated mice (▪), untreated mice (●), treated mice pre-injected with30-fold molar excess of PMPA (▾).

FIG. 13D. The effect of treatment with SK71 (2.0 μmol/kg) on percentageweight change in nu/nu mice previously treated with LNCaP cells in HCMatrigel. Treated mice (▪), untreated mice (●), treated micepre-injected with 30-fold molar excess of PMPA ( ).

FIG. 14A. The effect of treatment with SK77 (2.0 μmol/kg) on tumorvolume in nu/nu mice previously treated with LNCaP cells in HC Matrigel.Untreated mice (▪), treated mice (▾).

FIG. 14B. The effect of treatment with SK77 (2.0 μmol/kg) on percentageweight change in nu/nu mice previously treated with LNCaP cells in HCMatrigel. Untreated mice (▪), treated mice (▾).

FIG. 15A. MUPA 99mTc imaging agent conjugate (9-atom linker) with energyminimized computer model.

FIG. 15B. MUPA 99mTc imaging agent conjugate (syn-SK33, 14-atom linker)with energy minimized computer model.

DETAILED DESCRIPTION

Drug delivery conjugates are described herein where a PSMA bindingligand is attached to a releasable or non-releasable linker which isattached to a drug, therapeutic agent, diagnostic agent, or imagingagent.

Illustratively, the bivalent linkers described herein may be included inlinkers used to prepare PSMA-binding drug conjugates, PSMA-bindingimaging agent conjugates, and PSMA-binding diagnostic agent conjugatesof the following formulae:B-L-TAB-L-IAB-L-DAwhere B is a PSMA-binding moiety, including analogs or derivativesthereof, L is a linker, TA is a therapeutic agent, including analogs orderivatives thereof, IA is an imaging agent, including analogs orderivatives thereof, and DA is a diagnostic agent, including analogs orderivatives thereof. The linker L can comprise multiple bivalentlinkers, including the bivalent linkers described herein. It is also tobe understood that as used herein, TA collectively refers to therapeuticagents, and analogs and derivatives thereof, IA collectively refers toimaging agents, and analogs and derivatives thereof, and DA collectivelyrefers to diagnostic agents, and analogs and derivatives thereof.

The linker may also include one or more spacer linkers and optionallyadditional releasable linkers. The spacer and releasable linkers may beattached to each other in any order or combination. Similarly, the PSMAbinding ligand may be attached to a spacer linker or to a releasablelinker. Similarly, the drug, therapeutic agent, diagnostic agent, orimaging agent may be attached to a spacer linker or to a releasablelinker. Each of these components of the conjugates may be connectedthrough existing or additional heteroatoms on the targeting ligand,drug, therapeutic agent, diagnostic agent, imaging agent, releasable orspacer linker. Illustrative heteroatoms include nitrogen, oxygen,sulfur, and the formulae —(NHR¹NHR²)—, —SO—, —(SO₂)—, and —N(R³)O—,wherein R¹, R², and R³ are each independently selected from hydrogen,alkyl, heteroalkyl, heterocyclyl, aryl, heteroaryl, arylalkyl,heteroarylalkyl, and the like, each of which may be optionallysubstituted.

In one illustrative embodiment, compounds are described herein thatinclude linkers having predetermined length and diameter dimensions. Inone aspect, linkers are described herein that satisfy one or moreminimum length requirements, or a length requirement falling within apredetermined range. In another aspect, satisfaction of a minimum lengthrequirement may be understood to be determined by computer modeling ofthe extended conformations of linkers. In another aspect, satisfactionof a minimum length requirement may be understood to be determined byhaving a certain number of atoms, whether or not substituted, forming abackbone chain of atoms connecting the binding ligand (B) with the drug(D). In another embodiment, the backbone chain of atoms is cyclized withanother divalent fragment. In another aspect, linkers are describedherein that satisfy one or more maximum or minimum diameterrequirements. In another aspect, satisfaction of a maximum or minimumdiameter requirement may be understood to be determined by computermodeling of various conformations of linkers modeled as thespace-filling. CPK, or like configurations. In another aspect,satisfaction of a maximum or minimum diameter requirement may beunderstood to be apply to one or more selected portions of the linker,for example the portion of the linker proximal to the binding ligand(B), or the portion of the linker proximal to the drug (D), and thelike. In another aspect, linkers are described herein that satisfy oneor more chemical composition requirements, such as linkers that includeone or more polar groups that may positively interact with the one ormore Arg or Lys side-chain nitrogens and/or Asp or Glu side chainoxygens found in the funnel portion of PSMA. In one variation, linkersare described herein that satisfy one or more chemical compositionrequirements, such as linkers that include one or more non-polar groupsthat may positively interact with the one or more Tyr or Phe side-chaincarbons found in the funnel portion of PSMA.

In one embodiment, the atom-length of the linker is defined by thenumber of atoms separating the binding or targeting ligand B, or analogor derivative thereof, and the drug D, or analog or derivative thereof.Accordingly, in configurations where the binding ligand B, or analog orderivative thereof, is attached directly to the drug D, or analog orderivative thereof, the attachment is also termed herein as a “0-atom”linker. It is understood that such 0-atom linkers include theconfiguration wherein B and D are directly attached by removing ahydrogen atom from each attachment point on B and D, respectively. It isalso understood that such 0-atom linkers include the configurationwherein B and D are attached through an overlapping heteroatom byremoving a hydrogen atom from one of B or D, and a heteroatom functionalgroup, such as OH, SH, NH₂, and the like from the other of B or D. It isalso understood that such 0-atom linkers include the configurationwherein B and D are attached through a double bond, which may be formedby removing two hydrogen atoms from each attachment point on B and D,respectively, or whereby B and D are attached through one or moreoverlapping heteroatoms by removing two hydrogen atoms, one hydrogen andone heteroatom functional group, or two heteroatom functional groups,such as OH, SH, NH₂, and the like, from each of B or D. In addition, Band D may be attached through a double bond formed by removing a doublebonded heteroatom functional group, such as O, S, NH, and the like, fromone or both of B or D. It is also to be understood that such heteroatomfunctional groups include those attached to saturated carbon atoms,unsaturated carbon atoms (including carbonyl groups), and otherheteroatoms. Similarly, the length of linkers that are greater than 0atoms are defined in an analogous manner.

Accordingly, in another illustrative embodiment, linkers (L) aredescribed having a chain length of at least 7 atoms. In one variation,linkers (L) are described having a chain length of at least 14 atoms. Inanother variation, linkers (L) are described having a chain length inthe range from about 7 atoms to about 20 atoms. In another variation,linkers (L) are described having a chain length in the range from about14 atoms to about 24 atoms.

In another embodiment, the length of the linker (L) is defined bymeasuring the length of an extended conformation of the linker. Suchextended conformations may be measured in art-recognized computermodeling programs, such as PC Model 7 (MMX). Accordingly, in anotherillustrative embodiment, linkers are described having a chain length ofat least 15 Å, at least 20 Å, or at least 25 Å.

In another embodiment, linkers are described having at least onehydrophobic side chain group, such as an alkyl, cycloalkyl, aryl,arylalkyl, or like group, each of which is optionally substituted. Inone aspect, the hydrophobic group is included in the linker byincorporating one or more Phe or Tyr groups, including substitutedvariants thereof, and analogs and derivatives thereof, in the linkerchain. It is appreciated that such Phe and/or Tyr side chain groups mayform positive pi-pi (π-π) interactions with Tyr and Phe residues foundin the funnel of PSMA. In addition, it is appreciated that the presenceof large side chain branches, such as the arylalkyl groups found on Pheand Tyr may provide a level of conformational rigidity to the linker,thus limiting the degrees of freedom, and reducing coiling and promotingextended conformations of the linker. Without being bound by theory, itis appreciated that such entropy restrictions may increase the overallbinding energy of the bound conjugates described herein. In addition, itis appreciated that the rigidity increases that may be provided bysterically hindered side chains, such as Phe and Tyr described herein,may reduce or prevent coiling and interactions between the ligand andthe imaging agent. For example, computational energy minimization of arepresentative 9-atom and 14-atom linker (see, for example, FIGS. 15Aand 15B) shows that there are no intra-molecular interactions betweenthe ligand and the imaging agent. Moreover, the presence of side chainthe two Phe side chains appears to promote a more extended conformationin syn-SK33 (FIG. 15B) than in the aminohexanoic acid-containingconjugate (FIG. 15A)

It has been discovered herein that the funnel shaped tunnel leading tothe catalytic site or active site of PSMA imposes length, shape, and/orchemical composition requirements on the linker portion of conjugates ofPSMA binding ligands and therapeutic, diagnostic, and imaging agentsthat positively and negatively affect the interactions between PSMA andthose conjugates. Described herein are illustrative embodiments of thoseconjugates that include such length, shape, and/or chemical compositionrequirements on the linker. Such length, shape, and/or chemicalcomposition requirements were assessed using molecular modeling. Forexample, the space filling and surface model of the PSMA complex with(s)-2-(4-iodobenzensylphosphonomethyl)-pentanedioic [2-PMPA derivative]PDB ID code 2C6P were generated using PROTEIN EXPLORER. The PROTEINEXPLORER model verified the 20 Å deep funnel, and also showed diameterfeatures at various locations along the funnel that may be used todefine linkers having favorable structural features. In addition, themodel showed that close to the active site of PSMA, there are a highernumber of hydrophobic residues that may provide additional bindinginteractions when the corresponding functional groups are included inthe linker. Finally, the model showed the presence of three hydrophobicpockets that may provide additional binding interactions when thecorresponding functional groups are included in the linker.

In another illustrative embodiment, the following molecular models werecreated for a conjugate of MUPA and a tripeptide ^(99m)Tc imaging agentconnected by a 9-atom linker, as shown in FIG. 15A, and syn-SK33including a branched 14-atom linker, as shown in FIG. 15B. The modelswere created using PC Model 7 (MMX) with energy minimization, and usingthe following bond length parameters: C—C (sp³-sp³)=1.53 Å, C—C(sp³-sp²)=1.51 Å, C—N (sp³-N)=1.47 Å, C—N (sp²-N)=1.38 Å. Such modelsmay be used to calculate the length of the linker connecting the bindingligand (B) and the drug (D). In addition, such models may be modified tocreate extended conformations, and subsequently used to calculate thelength of the linker connecting the binding ligand (B) and the drug (D).

The first human PSMA gene was cloned from LNCaP cells and is reported tobe located in chromosome 11p11-12. In addition, there is a PSMA-likegene located at the loci 11q14.3. The crystal structure of PSMA has beenreported by two different groups at different resolutions, and eachshows that the active site contains two zinc atoms, confirming that PSMAis also considered a zinc metalloprotease. Davis et al. PNAS,102:5981-86, (2005) reported the crystal structure at low resolution(3.5 Å), while Mesters et al, The EMBO Journal, 1-10 (2006) reported thecrystal structure at higher resolution (2-2.2 Å), the disclosures ofwhich are incorporated herein by reference. The crystal structures showthat PSMA is a homodimer that contains a protease domain, an apicaldomain, a helical domain and a CPG2 dimerization domain. The proteasedomain of PSMA contains a binuclear zinc site, catalytic residues and asubstrate binding region including three arginine residues (alsoreferred to as a substrate binding arginine patch). In the crystalstructure, the two zinc ions in the active site are each ligated to anoxygen of phosphate, or to the phosphinate moiety of the inhibitor GPI18431 for the co-crystal structure. In the high resolution crystalstructures of the extracelluar domain, PSMA was co-crystallized withboth potent inhibitors, weak inhibitors, and glutamate at 2.0, 2.4, and2.2 Å, respectively. The high resolution crystal structure shows a 20 Ådeep funnel shaped tunnel leads to the catalytic site or active site ofPSMA. The funnel is lined with the side chains of a number of Arg andLys residues, Asp and Glu residues, and Tyr and Phe residues.

In another embodiment, the linker (L) is a chain of atoms selected fromC, N, O, S, Si, and P. The linker may have a wide variety of lengths,such as in the range from about 7 to about 100. The atoms used informing the linker may be combined in all chemically relevant ways, suchas chains of carbon atoms forming alkylene groups, chains of carbon andoxygen atoms forming polyoxyalkylene groups, chains of carbon andnitrogen atoms forming polyamines, and others. In addition, it is to beunderstood that the bonds connecting atoms in the chain may be eithersaturated or unsaturated, such that for example, alkanes, alkenes,alkynes, cycloalkanes, arylenes, imides, and the like may be divalentradicals that are included in the linker. In addition, it is to beunderstood that the atoms forming the linker may also be cyclized uponeach other to form divalent cyclic radicals in the linker. In each ofthe foregoing and other linkers described herein the chain forming thelinker may be substituted with a wide variety of groups.

In another embodiment, linkers (L) are described that include at leastone releasable linker. In one variation, linkers (L) are described thatinclude at least two releasable linkers. In another variation, linkers(L) are described that include at least one self-immolative linker. Inanother variation, linkers (L) are described that include at least onereleasable linker that is not a disulfide. In another embodiment,linkers (L) are described that do not include a releasable linker.

It is appreciated that releasable linkers may be used when the drug tobe delivered is advantageously liberated from the binding ligand-linkerconjugate so that the free drug will have the same or nearly the sameeffect at the target as it would when administered without the targetingprovided by the conjugates described herein. In another embodiment, thelinker L is a non-releasable linker. It is appreciated thatnon-releasable linkers may be used when the drug is advantageouslyretained by the binding ligand-linker conjugate, such as in imaging,diagnosing, uses of the conjugates described herein. It is to beunderstood that the choice of a releasable linker or a non-releasablelinker may be made independently for each application or configurationof the conjugates, without limiting the invention described herein. Itis to be further understood that the linkers L described herein comprisevarious atoms, chains of atoms, functional groups, and combinations offunctional groups. Where appropriate in the present disclosure, thelinker L may be referred to by the presence of spacer linkers,releasable linkers, and heteroatoms. However, such references are not tobe construed as limiting the definition of the linkers L describedherein.

The linker (L) comprising spacer and/or releasable linkers (i.e.,cleavable linkers) can be any biocompatible linker. The releasable orcleavable linker can be, for example, a linker susceptible to cleavageunder the reducing or oxidizing conditions present in or on cells, apH-sensitive linker that may be an acid-labile or base-labile linker, ora linker that is cleavable by biochemical or metabolic processes, suchas an enzyme-labile linker. In one embodiment, the spacer and/orreleasable linker comprises about 1 to about 30 atoms, or about 2 toabout 20 atoms. Lower molecular weight linkers (i.e., those having anapproximate molecular weight of about 30 to about 300) are alsodescribed. Precursors to such linkers may be selected to have eithernucleophilic or electrophilic functional groups, or both, optionally ina protected form with a readily cleavable protecting group to facilitatetheir use in synthesis of the intermediate species.

The term “releasable linker” as used herein refers to a linker thatincludes at least one bond that can be broken under physiologicalconditions (e.g., a pH-labile, acid-labile, oxidatively-labile, orenzyme-labile bond). The cleavable bond or bonds may be present in theinterior of a cleavable linker and/or at one or both ends of a cleavablelinker. It should be appreciated that such physiological conditionsresulting in bond breaking include standard chemical hydrolysisreactions that occur, for example, at physiological pH, or as a resultof compartmentalization into a cellular organelle such as an endosomehaving a lower pH than cytosolic pH. Illustratively, the bivalentlinkers described herein may undergo cleavage under other physiologicalor metabolic conditions, such as by the action of a glutathione mediatedmechanism. It is appreciated that the lability of the cleavable bond maybe adjusted by including functional groups or fragments within thebivalent linker L that are able to assist or facilitate such bondbreakage, also termed anchimeric assistance. The lability of thecleavable bond can also be adjusted by, for example, substitutionalchanges at or near the cleavable bond, such as including alpha branchingadjacent to a cleavable disulfide bond, increasing the hydrophobicity ofsubstituents on silicon in a moiety having a silicon-oxygen bond thatmay be hydrolyzed, homologating alkoxy groups that form part of a ketalor acetal that may be hydrolyzed, and the like. In addition, it isappreciated that additional functional groups or fragments may beincluded within the bivalent linker L that are able to assist orfacilitate additional fragmentation of the PSMA binding drug linkerconjugates after bond breaking of the releasable linker.

In another embodiment, the linker includes radicals that form one ormore spacer linkers and/or releasable linkers that are taken together toform the linkers described herein having certain length, diameter,and/or functional group requirements.

Another illustrative embodiment of the linkers described herein, includereleasable linkers that cleave under the conditions described herein bya chemical mechanism involving beta elimination. In one aspect, suchreleasable linkers include beta-thio, beta-hydroxy, and beta-aminosubstituted carboxylic acids and derivatives thereof, such as esters,amides, carbonates, carbamates, and ureas. In another aspect, suchreleasable linkers include 2- and 4-thioarylesters, carbamates, andcarbonates.

It is to be understood that releasable linkers may also be referred toby the functional groups they contain, illustratively such as disulfidegroups, ketal groups, and the like, as described herein. Accordingly, itis understood that a cleavable bond can connect two adjacent atomswithin the releasable linker and/or connect other linkers, or thebinding ligand B, or the therapeutic, diagnostic, or imaging agent D, asdescribed herein, at either or both ends of the releasable linker. Inthe case where a cleavable bond connects two adjacent atoms within thereleasable linker, following breakage of the bond, the releasable linkeris broken into two or more fragments. Alternatively, in the case where acleavable bond is between the releasable linker and another moiety, suchas an additional heteroatom, a spacer linker, another releasable linker,the drug D, or analog or derivative thereof, or the binding ligand B, oranalog or derivative thereof, following breakage of the bond, thereleasable linker is separated from the other moiety.

In another embodiment, the releasable and spacer linkers may be arrangedin such a way that subsequent to the cleavage of a bond in the bivalentlinker, released functional groups anchimerically assist the breakage orcleavage of additional bonds, as described above. An illustrativeembodiment of such a bivalent linker or portion thereof includescompounds having the formula:

where X is an heteroatom, such as nitrogen, oxygen, or sulfur, n is aninteger selected from 0, 1, 2, and 3, R is hydrogen, or a substituent,including a substituent capable of stabilizing a positive chargeinductively or by resonance on the aryl ring, such as alkoxy, and thelike, and the symbol (*) indicates points of attachment for additionalspacer or releasable linkers, or heteroatoms, forming the bivalentlinker, or alternatively for attachment of the drug, or analog orderivative thereof, or the vitamin, or analog or derivative thereof. Itis appreciated that other substituents may be present on the aryl ring,the benzyl carbon, the alkanoic acid, or the methylene bridge, includingbut not limited to hydroxy, alkyl, alkoxy, alkylthio, halo, and thelike. Assisted cleavage may include mechanisms involving benzyliumintermediates, benzyne intermediates, lactone cyclization, oxoniumintermediates, beta-elimination, and the like. It is further appreciatedthat, in addition to fragmentation subsequent to cleavage of thereleasable linker, the initial cleavage of the releasable linker may befacilitated by an anchimerically assisted mechanism.

In this embodiment, the hydroxyalkanoic acid, which may cyclize,facilitates cleavage of the methylene bridge, by for example an oxoniumion, and facilitates bond cleavage or subsequent fragmentation afterbond cleavage of the releasable linker. Alternatively, acid catalyzedoxonium ion-assisted cleavage of the methylene bridge may begin acascade of fragmentation of this illustrative bivalent linker, orfragment thereof. Alternatively, acid-catalyzed hydrolysis of thecarbamate may facilitate the beta elimination of the hydroxyalkanoicacid, which may cyclize, and facilitate cleavage of methylene bridge, byfor example an oxonium ion. It is appreciated that other chemicalmechanisms of bond breakage or cleavage under the metabolic,physiological, or cellular conditions described herein may initiate sucha cascade of fragmentation. It is appreciated that other chemicalmechanisms of bond breakage or cleavage under the metabolic,physiological, or cellular conditions described herein may initiate sucha cascade of fragmentation.

Illustrative mechanisms for cleavage of the bivalent linkers describedherein include the following 1,4 and 1,6 fragmentation mechanisms

where X is an exogenous or endogenous nucleophile, glutathione, orbioreducing agent, and the like, and either of Z or Z′ is a PSMA bindingligand, or a drug, therapeutic agent, diagnostic agent, or imagingagent, or either of Z or Z′ is a PSMA binding ligand, or a drug,therapeutic agent, diagnostic agent, or imaging agent connected throughother portions of the bivalent linker. It is to be understood thatalthough the above fragmentation mechanisms are depicted as concertedmechanisms, any number of discrete steps may take place to effect theultimate fragmentation of the bivalent linker to the final productsshown. For example, it is appreciated that the bond cleavage may alsooccur by acid catalyzed elimination of the carbamate moiety, which maybe anchimerically assisted by the stabilization provided by either thearyl group of the beta sulfur or disulfide illustrated in the aboveexamples. In those variations of this embodiment, the releasable linkeris the carbamate moiety. Alternatively, the fragmentation may beinitiated by a nucleophilic attack on the disulfide group, causingcleavage to form a thiolate. The thiolate may intermolecularly displacea carbonic acid or carbamic acid moiety and form the correspondingthiacyclopropane. In the case of the benzyl-containing bivalent linkers,following an illustrative breaking of the disulfide bond, the resultingphenyl thiolate may further fragment to release a carbonic acid orcarbamic acid moiety by forming a resonance stabilized intermediate. Inany of these cases, the releaseable nature of the illustrative bivalentlinkers described herein may be realized by whatever mechanism may berelevant to the chemical, metabolic, physiological, or biologicalconditions present.

Other illustrative mechanisms for bond cleavage of the releasable linkerinclude oxonium-assisted cleavage as follows:

where Z is the vitamin, or analog or derivative thereof, or the drug, oranalog or derivative thereof, or each is a vitamin or drug moiety inconjunction with other portions of the polyvalent linker, such as a drugor vitamin moiety including one or more spacer linkers and/or otherreleasable linkers. In this embodiment, acid-catalyzed elimination ofthe carbamate leads to the release of CO₂ and the nitrogen-containingmoiety attached to Z, and the formation of a benzyl cation, which may betrapped by water, or any other Lewis base.

In one embodiment, the releasable linker includes a disulfide.

In another embodiment, the releasable linker may be a divalent radicalcomprising alkyleneaziridin-1-yl, alkylenecarbonylaziridin-1-yl,carbonylalkylaziridin-1-yl, alkylenesulfoxylaziridin-1-yl,sulfoxylalkylaziridin-1-yl, sulfonylalkylaziridin-1-yl, oralkylenesulfonylaziridin-1-yl, wherein each of the releasable linkers isoptionally substituted with a substituent X², as defined below.

Additional illustrative releasable linkers include methylene,1-alkoxyalkylene, 1-alkoxycycloalkylene, 1-alkoxyalkylenecarbonyl,1-alkoxycycloalkylenecarbonyl, carbonylarylcarbonyl,carbonyl(carboxyaryl)carbonyl, carbonyl(biscarboxyaryl)carbonyl,haloalkylenecarbonyl, alkylene(dialkylsilyl), alkylene(alkylarylsilyl),alkylene(diarylsilyl), (dialkylsilyl)aryl, (alkylarylsilyl)aryl,(diarylsilyl)aryl, oxycarbonyloxy, oxycarbonyloxyalkyl, sulfonyloxy,oxysulfonylalkyl, iminoalkylidenyl, carbonylalkylideniminyl,iminocycloalkylidenyl, carbonylcycloalkylideniminyl, alkylenethio,alkylenearylthio, and carbonylalkylthio, wherein each of the releasablelinkers is optionally substituted with a substituent X², as definedbelow.

In the preceding embodiment, the releasable linker may include oxygen,and the releasable linkers can be methylene, 1-alkoxyalkylene,1-alkoxycycloalkylene, 1-alkoxyalkylenecarbonyl, and1-alkoxycycloalkylenecarbonyl, wherein each of the releasable linkers isoptionally substituted with a substituent X², as defined below, and thereleasable linker is bonded to the oxygen to form an acetal or ketal.Alternatively, the releasable linker may include oxygen, and thereleasable linker can be methylene, wherein the methylene is substitutedwith an optionally-substituted aryl, and the releasable linker is bondedto the oxygen to form an acetal or ketal. Further, the releasable linkermay include oxygen, and the releasable linker can be sulfonylalkyl, andthe releasable linker is bonded to the oxygen to form an alkylsulfonate.

In another embodiment of the above releasable linker embodiment, thereleasable linker may include nitrogen, and the releasable linkers canbe iminoalkylidenyl, carbonylalkylideniminyl, iminocycloalkylidenyl, andcarbonylcycloalkylideniminyl, wherein each of the releasable linkers isoptionally substituted with a substituent X², as defined below, and thereleasable linker is bonded to the nitrogen to form an hydrazone. In analternate configuration, the hydrazone may be acylated with a carboxylicacid derivative, an orthoformate derivative, or a carbamoyl derivativeto form various acylhydrazone releasable linkers.

Alternatively, the releasable linker may include oxygen, and thereleasable linkers can be alkylene(dialkylsilyl),alkylene(alkylarylsilyl), alkylene(diarylsilyl), (dialkylsilyl)aryl,(alkylarylsilyl)aryl, and (diarylsilyl)aryl, wherein each of thereleasable linkers is optionally substituted with a substituent X², asdefined below, and the releasable linker is bonded to the oxygen to forma silanol.

In the above releasable linker embodiment, the drug can include anitrogen atom, the releasable linker may include nitrogen, and thereleasable linkers can be carbonylarylcarbonyl,carbonyl(carboxyaryl)carbonyl, carbonyl(biscarboxyaryl)carbonyl, and thereleasable linker can be bonded to the heteroatom nitrogen to form anamide, and also bonded to the drug nitrogen to form an amide.

In the above releasable linker embodiment, the drug can include anoxygen atom, the releasable linker may include nitrogen, and thereleasable linkers can be carbonylarylcarbonyl,carbonyl(carboxyaryl)carbonyl, carbonyl(biscarboxyaryl)carbonyl, and thereleasable linker can form an amide, and also bonded to the drug oxygento form an ester.

The substituents X² can be alkyl, alkoxy, alkoxyalkyl, hydroxy,hydroxyalkyl, amino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl,halo, haloalkyl, sulfhydrylalkyl, alkylthioalkyl, aryl, substitutedaryl, arylalkyl, substituted arylalkyl, heteroaryl, substitutedheteroaryl, carboxy, carboxyalkyl, alkyl carboxylate, alkyl alkanoate,guanidinoalkyl, R⁴-carbonyl, R⁵-carbonylalkyl, R⁶-acylamino, andR⁷-acylaminoalkyl, wherein R⁴ and R⁵ are each independently selectedfrom amino acids, amino acid derivatives, and peptides, and wherein R⁶and R⁷ are each independently selected from amino acids, amino acidderivatives, and peptides. In this embodiment the releasable linker caninclude nitrogen, and the substituent X² and the releasable linker canform an heterocycle.

The heterocycles can be pyrrolidines, piperidines, oxazolidines,isoxazolidines, thiazolidines, isothiazolidines, pyrrolidinones,piperidinones, oxazolidinones, isoxazolidinones, thiazolidinones,isothiazolidinones, and succinimides.

In one embodiment, the polyvalent linkers described herein are orinclude compounds of the following formulae:

where n is an integer selected from 1 to about 4; R^(a) and R^(b) areeach independently selected from the group consisting of hydrogen andalkyl, including lower alkyl such as C₁-C₄ alkyl that are optionallybranched; or R^(a) and R^(b) are taken together with the attached carbonatom to form a carbocyclic ring; R is an optionally substituted alkylgroup, an optionally substituted acyl group, or a suitably selectednitrogen protecting group; and (*) indicates points of attachment forthe drug, vitamin, imaging agent, diagnostic agent, other polyvalentlinkers, or other parts of the conjugate.

In another embodiment, the polyvalent linkers described herein are orinclude compounds of the following formulae

where m is an integer selected from 1 to about 4; R is an optionallysubstituted alkyl group, an optionally substituted acyl group, or asuitably selected nitrogen protecting group; and (*) indicates points ofattachment for the drug, vitamin, imaging agent, diagnostic agent, otherpolyvalent linkers, or other parts of the conjugate.

In another embodiment, the polyvalent linkers described herein are orinclude compounds of the following formulae

where m is an integer selected from 1 to about 4; R is an optionallysubstituted alkyl group, an optionally substituted acyl group, or asuitably selected nitrogen protecting group; and (*) indicates points ofattachment for the drug, vitamin, imaging agent, diagnostic agent, otherpolyvalent linkers, or other parts of the conjugate.

In another embodiment, the linker L includes one or more spacer linkers.Such spacer linkers can be 1-alkylenesuccinimid-3-yl, optionallysubstituted with a substituent X¹, as defined below, and the releasablelinkers can be methylene, 1-alkoxyalkylene, 1-alkoxycycloalkylene,1-alkoxyalkylenecarbonyl, 1-alkoxycycloalkylenecarbonyl, wherein each ofthe releasable linkers is optionally substituted with a substituent X²,as defined below, and wherein the spacer linker and the releasablelinker are each bonded to the spacer linker to form asuccinimid-1-ylalkyl acetal or ketal.

The spacer linkers can be carbonyl, thionocarbonyl, alkylene,cycloalkylene, alkylenecycloalkyl, alkylenecarbonyl,cycloalkylenecarbonyl, carbonylalkylcarbonyl, 1-alkylenesuccinimid-3-yl,1-(carbonylalkyl)succinimid-3-yl, alkylenesulfoxyl, sulfonylalkyl,alkylenesulfoxylalkyl, alkylenesulfonylalkyl,carbonyltetrahydro-2H-pyranyl, carbonyltetrahydrofuranyl,1-(carbonyltetrahydro-2H-pyranyl)succinimid-3-yl, and1-(carbonyltetrahydrofuranyl)succinimid-3-yl, wherein each of the spacerlinkers is optionally substituted with a substituent X¹, as definedbelow. In this embodiment, the spacer linker may include an additionalnitrogen, and the spacer linkers can be alkylenecarbonyl,cycloalkylenecarbonyl, carbonylalkylcarbonyl,1-(carbonylalkyl)succinimid-3-yl, wherein each of the spacer linkers isoptionally substituted with a substituent X¹, as defined below, and thespacer linker is bonded to the nitrogen to form an amide. Alternatively,the spacer linker may include an additional sulfur, and the spacerlinkers can be alkylene and cycloalkylene, wherein each of the spacerlinkers is optionally substituted with carboxy, and the spacer linker isbonded to the sulfur to form a thiol. In another embodiment, the spacerlinker can include sulfur, and the spacer linkers can be1-alkylenesuccinimid-3-yl and 1-(carbonylalkyl)succinimid-3-yl, and thespacer linker is bonded to the sulfur to form a succinimid-3-ylthiol.

In an alternative to the above-described embodiments, the spacer linkercan include nitrogen, and the releasable linker can be a divalentradical comprising alkyleneaziridin-1-yl, carbonylalkylaziridin-1-yl,sulfoxylalkylaziridin-1-yl, or sulfonylalkylaziridin-1-yl, wherein eachof the releasable linkers is optionally substituted with a substituentX², as defined below. In this alternative embodiment, the spacer linkerscan be carbonyl, thionocarbonyl, alkylenecarbonyl,cycloalkylenecarbonyl, carbonylalkylcarbonyl,1-(carbonylalkyl)succinimid-3-yl, wherein each of the spacer linkers isoptionally substituted with a substituent X¹, as defined below, andwherein the spacer linker is bonded to the releasable linker to form anaziridine amide.

The substituents X¹ can be alkyl, alkoxy, alkoxyalkyl, hydroxy,hydroxyalkyl, amino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl,halo, haloalkyl, sulfhydrylalkyl, alkylthioalkyl, aryl, substitutedaryl, arylalkyl, substituted arylalkyl, heteroaryl, substitutedheteroaryl, carboxy, carboxyalkyl, alkyl carboxylate, alkyl alkanoate,guanidinoalkyl, R⁴-carbonyl, R⁵-carbonylalkyl, R⁶-acylamino, andR⁷-acylaminoalkyl, wherein R⁴ and R⁵ are each independently selectedfrom amino acids, amino acid derivatives, and peptides, and wherein R⁶and R⁷ are each independently selected from amino acids, amino acidderivatives, and peptides. In this embodiment the spacer linker caninclude nitrogen, and the substituent X¹ and the spacer linker to whichthey are bound to form an heterocycle.

Additional illustrative spacer linkers includealkylene-amino-alkylenecarbonyl,alkylene-thio-(carbonylalkylsuccinimid-3-yl), and the like, as furtherillustrated by the following formulae:

where the integers x and y are 1, 2, 3, 4, or 5:

In another embodiment, linkers that include hydrophilic regions are alsodescribed. In one aspect, the hydrophilic region of the linker formspart or all of a spacer linker included in the conjugates describedherein. Illustrative hydrophilic spacer linkers are described in PCTinternational application serial No. PCT/US2008/068093, filed Jun. 25,2008, the disclosure of which is incorporated herein by reference.

The term “cycloalkyl” as used herein includes molecular fragments orradicals comprising a bivalent chain of carbon atoms, a portion of whichforms a ring. It is to be understood that the term cycloalkyl as usedherein includes fragments and radicals attached at either ring atoms ornon-ring atoms, such as, such as cyclopropyl, cyclohexyl,3-ethylcyclopent-1-yl, cyclopropylethyl, cyclohexylmethyl, and the like.

The term “cycloalkylene” as used herein includes molecular fragments orradicals comprising a bivalent chain of carbon atoms, a portion of whichforms a ring. It is to be understood that the term cycloalkyl as usedherein includes fragments and radicals attached at either ring atoms ornon-ring atoms, such as cycloprop-1,1-diyl, cycloprop-1,2-diyl,cyclohex-1,4-diyl, 3-ethylcyclopent-1,2-diyl, 1-methylenecyclohex-4-yl,and the like.

The terms “heteroalkyl” and “heteroalkylene” as used herein includesmolecular fragments or radicals comprising monovalent and divalent,respectively, groups that are formed from a linear or branched chain ofcarbon atoms and heteroatoms, wherein the heteroatoms are selected fromnitrogen, oxygen, and sulfur, such as alkoxyalkyl, alkyleneoxyalkyl,aminoalkyl, alkylaminoalkyl, alkyleneaminoalkyl, alkylthioalkyl,alkylenethioalkyl, alkoxyalkylaminoalkyl, alkyl aminoalkoxyalkyl,alkyleneoxyalkylaminoalkyl, and the like.

The term “heterocyclyl” as used herein includes molecular fragments orradicals comprising a monovalent chain of carbon atoms and heteroatoms,wherein the heteroatoms are selected from nitrogen, oxygen, and sulfur,a portion of which, including at least one heteroatom, form a ring, suchas aziridine, pyrrolidine, oxazolidine, 3-methoxypyrrolidine,3-methylpiperazine, and the like. Accordingly, as used herein,heterocyclyl includes alkylheterocyclyl, heteroalkylheterocyclyl,heterocyclylalkyl, heterocyclylheteroalkyl, and the like. It is to beunderstood that the term heterocyclyl as used herein includes fragmentsand radicals attached at either ring atoms or non-ring atoms, such astetrahydrofuran-2-yl, piperidin-1-yl, piperidin-4-yl, piperazin-1-yl,morpholin 1 yl, tetrahydrofuran-2-ylmethyl, piperidin-1-ylethyl,piperidin-4-ylmethyl, piperazin-1-ylpropyl, morpholin-1-ylethyl, and thelike.

The term “aryl” as used herein includes molecular fragments or radicalscomprising an aromatic mono or polycyclic ring of carbon atoms, such asphenyl, naphthyl, and the like.

The term “heteroaryl” as used herein includes molecular fragments orradicals comprising an aromatic mono or polycyclic ring of carbon atomsand at least one heteroatom selected from nitrogen, oxygen, and sulfur,such as pyridinyl, pyrimidinyl, indolyl, benzoxazolyl, and the like.

The term “substituted aryl” or “substituted heteroaryl” as used hereinincludes molecular fragments or radicals comprising aryl or heteroarylsubstituted with one or more substituents, such as alkyl, heteroalkyl,halo, hydroxy, amino, alkyl or dialkylamino, alkoxy, alkylsulfonyl,aminosulfonyl, carboxylate, alkoxycarbonyl, aminocarbonyl, cyano, nitro,and the like. It is to be understood that the alkyl groups in suchsubstituents may be optionally substituted with halo.

The term “iminoalkylidenyl” as used herein includes molecular fragmentsor radicals comprising a divalent radical containing alkylene as definedherein and a nitrogen atom, where the terminal carbon of the alkylene isdouble-bonded to the nitrogen atom, such as the formulae —(CH)═N—,—(CH₂)₂(CH)═N—, —CH₂C(Me)═N—, and the like.

The term “amino acid” as used herein includes molecular fragments orradicals comprising an aminoalkylcarboxylate, where the alkyl radical isoptionally substituted with alkyl, hydroxy alkyl, sulfhydrylalkyl,aminoalkyl, carboxyalkyl, and the like, including groups correspondingto the naturally occurring amino acids, such as serine, cysteine,methionine, aspartic acid, glutamic acid, and the like.

For example, in one embodiment, amino acid is a divalent radical havingthe general formula:—N(R)—(CR′R″)_(q)—C(O)—where R is hydrogen, alkyl, acyl, or a suitable nitrogen protectinggroup, R′ and R″ are hydrogen or a substituent, each of which isindependently selected in each occurrence, and q is an integer such as1, 2, 3, 4, or 5. Illustratively. R′ and/or R″ independently correspondto, but are not limited to, hydrogen or the side chains present onnaturally occurring amino acids, such as methyl, benzyl, hydroxymethyl,thiomethyl, carboxyl, carboxylmethyl, guanidinopropyl, and the like, andderivatives and protected derivatives thereof. The above describedformula includes all stereoisomeric variations. For example, the aminoacid may be selected from asparagine, aspartic acid, cysteine, glutamicacid, lysine, glutamine, arginine, serine, ornitine, threonine, and thelike. In one variation, the amino acid may be selected fromphenylalanine, tyrosine, and the like, derivatives thereof, andsubstituted variants thereof.

The terms “arylalkyl” and “heteroarylalkyl” as used herein includesmolecular fragments or radicals comprising aryl and heteroaryl,respectively, as defined herein substituted with a linear or branchedalkylene group, such as benzyl, phenethyl, α-methylbenzyl, picolinyl,pyrimidinylethyl, and the like.

It is to be understood that the above-described terms can be combined togenerate chemically-relevant groups, such as “haloalkoxyalkyl” referringto for example trifluoromethyloxyethyl,1,2-difluoro-2-chloroeth-1-yloxypropyl, and the like.

The term “amino acid derivative” as used herein refers generally toaminoalkylcarboxylate, where the amino radical or the carboxylateradical are each optionally substituted with alkyl, carboxylalkyl,alkylamino, and the like, or optionally protected; and the interveningdivalent alkyl fragment is optionally substituted with alkyl, hydroxyalkyl, sulfhydrylalkyl, aminoalkyl, carboxyalkyl, and the like,including groups corresponding to the side chains found in naturallyoccurring amino acids, such as are found in serine, cysteine,methionine, aspartic acid, glutamic acid, and the like.

The term “peptide” as used herein includes molecular fragments orradicals comprising a series of amino acids and amino acid analogs andderivatives covalently linked one to the other by amide bonds.

In another embodiment, the bivalent linker comprises a spacer linker anda releasable linker taken together to form3-thiosuccinimid-1-ylalkyloxymethyloxy, where the methyl is optionallysubstituted with alkyl or substituted aryl.

In another embodiment, the bivalent linker comprises a spacer linker anda releasable linker taken together to form3-thiosuccinimid-1-ylalkylcarbonyl, where the carbonyl forms anacylaziridine with the drug, or analog or derivative thereof.

In another embodiment, the bivalent linker comprises an a spacer linkerand a releasable linker taken together to form 1-alkoxycycloalkylenoxy.

In another embodiment, the bivalent linker comprises a spacer linker anda releasable linker taken together to formalkyleneaminocarbonyl(dicarboxylarylene)carboxylate.

In another embodiment, the bivalent linker comprises a releasablelinker, a spacer linker, and a releasable linker taken together to formdithioalkylcarbonylhydrazide, where the hydrazide forms an hydrazonewith the drug, or analog or derivative thereof.

In another embodiment, the bivalent linker comprises a spacer linker anda releasable linker taken together to form3-thiosuccinimid-1-ylalkylcarbonylhydrazide, where the hydrazide formsan hydrazone with the drug, or analog or derivative thereof.

In another embodiment, the bivalent linker comprises a spacer linker anda releasable linker taken together to form3-thioalkylsulfonylalkyl(disubstituted silyl)oxy, where thedisubstituted silyl is substituted with alkyl or optionally substitutedaryl.

In another embodiment, the bivalent linker comprises a plurality ofspacer linkers selected from the group consisting of the naturallyoccurring amino acids and stereoisomers thereof.

In another embodiment, the bivalent linker comprises a releasablelinker, a spacer linker, and a releasable linker taken together to form3-dithioalkyloxycarbonyl, where the carbonyl forms a carbonate with thedrug, or analog or derivative thereof.

In another embodiment, the bivalent linker comprises a releasablelinker, a spacer linker, and a releasable linker taken together to form3-dithioarylalkyloxycarbonyl, where the carbonyl forms a carbonate withthe drug, or analog or derivative thereof, and the aryl is optionallysubstituted.

In another embodiment, the bivalent linker comprises a spacer linker anda releasable linker taken together to form3-thiosuccinimid-1-ylalkyloxyalkyloxyalkylidene, where the alkylideneforms an hydrazone with the drug, or analog or derivative thereof, eachalkyl is independently selected, and the oxyalkyloxy is optionallysubstituted with alkyl or optionally substituted aryl.

In another embodiment, the bivalent linker comprises a releasablelinker, a spacer linker, and a releasable linker taken together to form3-dithioalkyloxycarbonylhydrazide.

In another embodiment, the bivalent linker comprises a releasablelinker, a spacer linker, and a releasable linker taken together to form3-dithioalkylamino, where the amino forms a vinylogous amide with thedrug, or analog or derivative thereof.

In another embodiment, the bivalent linker comprises a releasablelinker, a spacer linker, and a releasable linker taken together to form3-dithioalkylamino, where the amino forms a vinylogous amide with thedrug, or analog or derivative thereof, and the alkyl is ethyl.

In another embodiment, the bivalent linker comprises a releasablelinker, a spacer linker, and a releasable linker taken together to form3-dithioalkylaminocarbonyl, where the carbonyl forms a carbamate withthe drug, or analog or derivative thereof.

In another embodiment, the bivalent linker comprises a releasablelinker, a spacer linker, and a releasable linker taken together to form3-dithioalkylaminocarbonyl, where the carbonyl forms a carbamate withthe drug, or analog or derivative thereof, and the alkyl is ethyl.

In another embodiment, the bivalent linker comprises a releasablelinker, a spacer linker, and a releasable linker taken together to form3-dithioarylalkyloxycarbonyl, where the carbonyl forms a carbamate or acarbamoylaziridine with the drug, or analog or derivative thereof.

In another embodiment, the polyvalent linker includes spacer linkers andreleasable linkers connected to form a polyvalent3-thiosuccinimid-1-ylalkyloxymethyloxy group, illustrated by thefollowing formula

where n is an integer from 1 to 6, the alkyl group is optionallysubstituted, and the methyl is optionally substituted with an additionalalkyl or optionally substituted aryl group, each of which is representedby an independently selected group R. The (*) symbols indicate points ofattachment of the polyvalent linker fragment to other parts of theconjugates described herein.

In another embodiment, the polyvalent linker includes spacer linkers andreleasable linkers connected to form a polyvalent3-thiosuccinimid-1-ylalkylcarbonyl group, illustrated by the followingformula

where n is an integer from 1 to 6, and the alkyl group is optionallysubstituted. The (*) symbols indicate points of attachment of thepolyvalent linker fragment to other parts of the conjugates describedherein. In another embodiment, the polyvalent linker includes spacerlinkers and releasable linkers connected to form a polyvalent3-thioalkylsulfonylalkyl(disubstituted silyl)oxy group, where thedisubstituted silyl is substituted with alkyl and/or optionallysubstituted aryl groups.

In another embodiment, the polyvalent linker includes spacer linkers andreleasable linkers connected to form a polyvalentdithioalkylcarbonylhydrazide group, or a polyvalent3-thiosuccinimid-1-ylalkylcarbonylhydrazide, illustrated by thefollowing formulae

where n is an integer from 1 to 6, the alkyl group is optionallysubstituted, and the hydrazide forms an hydrazone with (B), (D), oranother part of the polyvalent linker (L). The (*) symbols indicatepoints of attachment of the polyvalent linker fragment to other parts ofthe conjugates described herein.

In another embodiment, the polyvalent linker includes spacer linkers andreleasable linkers connected to form a polyvalent3-thiosuccinimid-1-ylalkyloxyalkyloxyalkylidene group, illustrated bythe following formula

where each n is an independently selected integer from 1 to 6, eachalkyl group independently selected and is optionally substituted, suchas with alkyl or optionally substituted aryl, and where the alkylideneforms an hydrazone with (B), (D), or another part of the polyvalentlinker (L). The (*) symbols indicate points of attachment of thepolyvalent linker fragment to other parts of the conjugates describedherein.

Additional illustrative linkers are described in WO 2006/012527, thedisclosure of which is incorporated herein by reference. Additionallinkers are described in the following Table, where the (*) atom is thepoint of attachment of additional spacer or releasable linkers, thedrug, and/or the binding ligand.

Illustrative Releasable Linkers

Each of the spacer and releasable linkers described herein is bivalent.In addition, the connections between spacer linkers, releasable linkers,drugs D and ligands B may occur at any atom found in the various spacerlinkers, releasable linkers, drugs D, and ligands B.

The drug can include a nitrogen atom, and the releasable linker can behaloalkylenecarbonyl, optionally substituted with a substituent X², andthe releasable linker is bonded to the drug nitrogen to form an amide.

The drug can include an oxygen atom, and the releasable linker can behaloalkylenecarbonyl, optionally substituted with a substituent X², andthe releasable linker is bonded to the drug oxygen to form an ester.

The drug can include a double-bonded nitrogen atom, and in thisembodiment, the releasable linkers can be alkylenecarbonylamino and1-(alkylenecarbonylamino)succinimid-3-yl, and the releasable linker canbe bonded to the drug nitrogen to form an hydrazone.

The drug can include a sulfur atom, and in this embodiment, thereleasable linkers can be alkylenethio and carbonylalkylthio, and thereleasable linker can be bonded to the drug sulfur to form a disulfide.

In another embodiment, the binding or targeting ligand capable ofbinding or targeting PSMA is a phosphoric, phosphonic, or phosphinicacid or derivative thereof. In one aspect, the phosphoric, phosphonic,or phosphinic acid or derivative thereof includes one or more carboxylicacid groups. In another aspect, the phosphoric, phosphonic, orphosphinic acid or derivative thereof includes one or more thiol groupsor derivatives thereof. In another aspect, the phosphoric, phosphonic,or phosphinic acid or derivative thereof includes one or more carboxylicacid bioisosteres, such as an optionally substituted tetrazole, and thelike.

In another embodiment, the PSMA ligand is a derivative of pentanedioicacid. Illustratively, the pentanedioic acid derivative is a compound ofthe formula:

wherein X is RP(O)(OH)CH₂— (see, e.g., U.S. Pat. No. 5,968,915incorporated herein by reference); RP(O)(OH)N(R′)— (see, e.g., U.S. Pat.No. 5,863,536 incorporated herein by reference); RP(O)(OH)O— (see, e.g.,U.S. Pat. No. 5,795,877 incorporated herein by reference); RN(OH)C(O)Y—or RC(O)NH(OH)Y, wherein Y is —CR₁R₂—, —NR₃— or —O— (see, e.g., U.S.Pat. No. 5,962,521 incorporated herein by reference); RS(O)Y, RSO₂Y, orRS(O)(NH)Y, wherein Y is —CR₁R₂—, —NR₃— or —O— (see, e.g., U.S. Pat. No.5,902,817 incorporated herein by reference); and RS-alkyl, wherein R isfor example hydrogen, alkyl, aryl, or arylalkyl, each of which may beoptionally substituted (see, e.g., J. Med. Chem. 46:1989-1996 (2003)incorporated herein by reference).

In each of the foregoing formulae, R, R₁, R₂, and R₃ are eachindependently selected from hydrogen, C₁-C₉ straight or branched chainalkyl, C₂-C₉ straight or branched chain alkenyl, C₃-C₈ cycloalkyl, C₅-C₇cycloalkenyl, and aryl. In addition, in each case, each of R, R₁, R₂,and R₃ may be optionally substituted, such as with one or more groupsselected from C₃-C₈ cycloalkyl, C₅-C₇ cycloalkenyl, halo, hydroxy,nitro, trifluoromethyl, C₁-C₆ straight or branched chain alkyl, C₂-C₆straight or branched chain alkenyl, C₁-C₄ alkoxy, C₂-C₄ alkenyloxy,phenoxy, benzyloxy, amino, aryl. In one aspect, aryl is selected from1-naphthyl, 2-naphthyl, 2-indolyl, 3-indolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, benzyl, andphenyl, and in each case aryl may be optionally substituted with one ormore, illustratively with one to three, groups selected from halo,hydroxy, nitro, trifluoromethyl, C₁-C₆ straight or branched chain alkyl,C₂-C₆ straight or branched chain alkenyl, C₁-C₄ alkoxy, C₂-C₄alkenyloxy, phenoxy, benzyloxy, and amino. In one variation of each ofthe above formulae, R is not hydrogen.

Illustrative PSMA ligands described in U.S. Pat. No. 5,968,915 include2-[[methylhydroxyphosphinyl]methyl]pentanedioic acid;2-[[ethylhydroxyphosphinyl]methyl]pentanedioic acid;2-[[propylhydroxyphosphinyl]methyl]pentanedioic acid;2-[[butylhydroxyphosphinyl]methyl]pentanedioic acid;2-[[cyclohexylhydroxyphosphinyl]methyl]pentanedioic acid;2-[[phenylhydroxyphosphinyl]methyl]pentanedioic acid;2-[[2-(tetrahydrofuranyl)hydroxyphosphinyl]methyl]pentanedioic acid;2-[[(2-tetrahydropyranyl)hydroxyphosphinyl]methyl]pentanedioic acid;2-[[((4-pyridyl)methyl)hydroxyphosphinyl]methyl]pentanedioic acid;2-[[(2-pyridyl)methyl)hydroxyphosphinyl]methyl]pentanedioic acid;2-[[(phenylmethyl)hydroxyphosphinyl]methyl] pentanedioic acid;2-[[((2-phenylethyl)methyl)hydroxyphosphinyl]methyl]pentanedioic acid;2-[[((3-phenylpropyl)methyl)hydroxyphosphinyl]methyl]pentanedioic acid;2-[[((3-phenylbutyl)methyl)hydroxyphosphinyl]methyl]pentanedioic acid;2-[[((2-phenylbutyl)methyl)hydroxyphosphinyl]methyl]pentanedioic acid;2-[[(4-phenylbutyl)hydroxyphosphinyl]methyl]pentanedioic acid; and2-[[(aminomethyl)hydroxyphosphinyl]methyl]pentanedioic acid.

Illustrative PSMA ligands described in U.S. Pat. No. 5,863,536 includeN-[methylhydroxyphosphinyl]glutamic acid;N-[ethylhydroxyphosphinyl]glutamic acid;N-[propylhydroxyphosphinyl]glutamic acid;N-[butylhydroxyphosphinyl]glutamic acid;N-[phenylhydroxyphosphinyl]glutamic acid;N-[(phenylinethyl)hydroxyphosphinyl]glutamic acid;N-[((2-phenylethyl)methyl)hydroxyphosphinyl]glutamic acid; andN-methyl-N-[phenylhydroxyphosphinyl]glutamic acid.

Illustrative PSMA ligands described in U.S. Pat. No. 5,795,877 include2-[[methylhydroxyphosphinyl]oxy]pentanedioic acid;2-[[ethylhydroxyphosphinyl]oxy]pentanedioic acid;2-[[propylhydroxyphosphinyl]oxy]pentanedioic acid;2-[[butylhydroxyphosphinyl]oxy]pentanedioic acid;2-[[phenylhydroxyphosphinyl]oxy]pentanedioic acid;2-[[((4-pyridyl)methyl)hydroxyphosphinyl]oxy]pentanedioic acid;2-[[((2-pyridyl)methyl)hydroxyphosphinyl]oxy]pentanedioic acid;2-[[(phenylmethyl)hydroxyphosphinyl]oxy]pentanedioic acid; and2[[((2-phenylethyl)methyl)hydroxyphosphinyl]oxy]pentanedioic acid.

Illustrative PSMA ligands described in U.S. Pat. No. 5,962,521 include2-[[(N-hydroxy)carbamoyl]methyl]pentanedioic acid;2-[[(N-hydroxy-N-methyl)carbamoyl]methyl]pentanedioic acid;2-[[(N-butyl-N-hydroxy) carbamoyl]methyl]pentanedioic acid;2-[[(N-benzyl-N-hydroxy)carbamoyl]methyl]pentanedioic acid;2-[[(N-hydroxy-N-phenyl)carbamoyl]methyl]pentanedioic acid;2-[[(N-hydroxy-N-2-phenylethyl)carbamoyl]methyl]pentanedioic acid;2-[[(N-ethyl-N-hydroxy) carbamoyl]methyl]pentanedioic acid;2-[[(N-hydroxy-N-propyl)carbamoyl]methyl]pentanedioic acid;2-[[(N-hydroxy-N-3-phenylpropyl)carbamoyl]methyl]pentanedioic acid;2-[[(N-hydroxy-N-4-pyridyl) carbamoyl]methyl]pentanedioic acid;2-[[(N-hydroxy)carboxamido]methyl]pentanedioic acid; 2-[[N-hydroxy(methyl) carboxamido]methyl]pentanedioic acid; 2-[[N-hydroxy (benzyl)carboxamido]methyl]pentanedioic acid;2-[[N-hydroxy(phenyl)carboxamido]methyl]pentanedioic acid;2-[[N-hydroxy(2-phenylethyl)carboxamido]methyl]pentanedioic acid;2-[[N-hydroxy(ethyl)carboxamido]methyl]pentanedioic acid;2-[[N-hydroxy(propyl) carboxamido]methyl]pentanedioic acid;2-[[N-hydroxy (3-phenylpropyl) carboxamido]methyl]pentanedioic acid; and2-[[N-hydroxy(4-pyridyl)carboxamido]methyl]pentanedioic acid.

Illustrative PSMA ligands described in U.S. Pat. No. 5,902,817 include2-[(sulfinyl)methyl]pentanedioic acid;2-[(methylsulfinyl)methyl]pentanedioic acid;2-[(ethylsulfinyl)methyl]pentanedioic acid;2-[(propylsulfinyl)methyl]pentanedioic acid;2-[(butylsulfinyl)methyl]pentanedioic acid;2-[(phenylsulfinyl]methyl]pentanedioic acid;2-[[(2-phenylethyl)sulfinyl]methyl]pentanedioic acid;2-[[(3-phenylpropyl)sulfinyl]methyl]pentanedioic acid;2-[[(4-pyridyl)sulfinyl]methyl]pentanedioic acid;2-[(benzylsulfinyl)methyl]pentanedioic acid;2-[(sulfonyl)methyl]pentanedioic acid;2-[(methylsulfonyl)methyl]pentanedioic acid;2-[(ethylsulfonyl)methyl]pentanedioic acid;2-[(propylsulfonyl)methyl]pentanedioic acid;2-[(butylsulfonyl)methyl]pentanedioic acid;2-[(phenylsulfonyl]methyl]pentanedioic acid;2-[[(2-phenylethyl)sulfonyl]methyl]pentanedioic acid;2-[[(3-phenylpropyl)sulfonyl]methyl]pentanedioic acid; 2-[[(4-pyridyl)sulfonyl]methyl]pentanedioic acid;2-[(benzylsulfonyl)methyl]pentanedioic acid;2-[(sulfoximinyl)methyl]pentanedioic acid;2-[(methylsulfoximinyl)methyl]pentanedioic acid;2-[(ethylsulfoximinyl)methyl]pentanedioic acid;2-[(propylsulfoximinyl)methyl]pentanedioic acid;2-[(butylsulfoximinyl)methyl]pentanedioic acid;2-[(phenylsulfoximinyl]methyl]pentanedioic acid;2-[[(2-phenylethyl)sulfoximinyl]methyl]pentanedioic acid;2-[[(3-phenylpropyl) sulfoximinyl]methyl]pentanedioic acid;2-[[(4-pyridyl)sulfoximinyl]methyl]pentanedioic acid; and2-[(benzylsulfoximinyemethyl]pentanedioic acid.

Pentanedioic acid derivatives described herein have been reported tohave high binding affinity at PSMA, including but not limited to thefollowing phosphonic and phosphinic acid derivatives

with the dissociation constants (K_(i) values) shown for the E-I complex(see, Current Medicinal Chem. 8:949-0.957 (2001); Silverman, “TheOrganic Chemistry of Drug Design and Drug Action,” Elsevier AcademicPress (2^(nd) Ed. 2003), the disclosures of which are incorporatedherein by reference);

In another illustrative embodiment, the pentanedioic acid derivativeincludes a thiol group, such as compounds of the following formulae:

with the inhibition constants (IC₅₀ values) shown for the E-I complex.

In another embodiment, the PSMA ligand is a urea of two amino acids. Inone aspect, the amino acids include one or more additional carboxylicacids. In another aspect, the amino acids include one or more additionalphosphoric, phosphonic, phosphinic, sulfinic, sulfonic, or boronicacids. In another aspect, the amino acids include one or more thiolgroups or derivatives thereof. In another aspect, the amino acidsincludes one or more carboxylic acid bioisosteres, such as tetrazolesand the like.

In another embodiment, the PSMA ligand is a aminocarbonyl derivative ofpentanedioic acid. Illustratively, the aminocarbonylpentanedioic acidderivative is a compound of the formula:

wherein R¹ and R² are each selected from hydrogen, optionallysubstituted carboxylic acids, such as thiolacetic acids, thiolpropionicacids, and the like; malonic acids, succinic acids, glutamic acids,adipic acids, and the like; and others. Illustrativeaminocarbonylpentanedioic acid derivatives are described in J. Med.Chem. 44:298-301 (2001) and J. Med. Chem. 47:1729-38 (2004), thedisclosures of which are incorporated herein by reference.

In another embodiment, the PSMA ligand is a compound of the formula:

R¹ K_(i) (nM)

6.9 (R = H) 29 (R = tert-Bu)

12 (R = H) 3.0 (R = OH)

 8

0.9 (R = H) 5.3 (R = CH₂CH₂CN)

2.1 (R = H) 5.9 (R = OH)

335

It is appreciated that the urea compounds described herein may also beadvantageous in the preparation of the ligands also described herein dueto the sub-nanomolar potency, water solubility, and/or long termstability of these compounds. The urea compounds described herein maygenerally be prepared from commercially available starting materials asdescribed herein.

It is appreciated that in each of the above illustrative pentanedioicacid compounds and urea compounds, there is at least one asymmetriccarbon atom. Accordingly, the above illustrative formulae are intendedto refer both individually and collectively to all stereoisomers as pureenantiomers, or mixtures of enantiomers and/or diastereomers, includingbut not limited to racemic mixtures, mixtures that include one epimer ata first asymmetric carbon but allow mixtures at other asymmetriccarbons, including racemic mixtures, and the like.

In another illustrative embodiment, the binding agent is a urea of anamino dicarboxylic acid, such as aspartic acid, glutamic acid, and thelike, and another amino dicarboxylic acid, or an analog thereof, such asa binding agent of the formulae

wherein Q is a an amino dicarboxylic acid, such as aspartic acid,glutamic acid, or an analog thereof, n and m are each selected from aninteger between 1 and about 6, and (*) represents the point ofattachment for the linker L.

In another embodiment, the PSMA ligand includes at least four carboxylicacid groups, or at least three free carboxylic acid groups after thePSMA ligand is conjugated to the agent or linker. It is understood thatas described herein, carboxylic acid groups on the PSMA ligand includebioisosteres of carboxylic acids.

Illustratively, the PSMA ligand is a compound of the formulae:

In another embodiment, the PSMA ligand is2-[3-(1-Carboxy-2-mercapto-ethyl)-ureido]-pentanedioic acid (MUPA) or2-[3-(1,3-Dicarboxy-propyl)-ureido]-pentanedioic acid (DUPA)

Other illustrative examples of PSMA ligands include peptide analogs suchas quisqualic acid, aspartate glutamate (Asp-Glu), Glu-Glu, Gly-Glu,y-Glu-Glu, beta-N-acetyl-L-aspartate-L-glutamate (β-NAAG), and the like.

In another illustrative embodiment, the binding agent is a urea of anamino dicarboxylic acid, such as aspartic acid, glutamic acid, and thelike, and another amino dicarboxylic acid, or an analog thereof, and thelinker is peptide of amino acids, including naturally occurring andnon-naturally occurring amino acids. In one embodiment, the linker is apeptide comprising amino acids selected from Glu, Asp, Phe, Cys,beta-amino Ala, and aminoalkylcarboxylic acids, such as Gly, beta Ala,amino valeric acid, amino caproic acid, and the like. In anotherembodiment, the linker is a peptide consisting of amino acids selectedfrom Glu, Asp, Phe, Cys, beta-amino Ala, and aminoalkylcarboxylic acids,such as Gly, beta Ala, amino valeric acid, amino caproic acid, and thelike. In another embodiment, the linker is a peptide comprising at leastone Phe. In variations, the linker is a peptide comprising at least twoPhe residues, or at least three Phe residues. In another embodiment, thelinker is a peptide comprising Glu-Phe or a dipeptide of anaminoalkylcarboxylic acid and Phe. In another embodiment, the linker isa peptide comprising Glu-Phe-Phe or a tripeptide of anaminoalkylcarboxylic acid and two Phe residues. In another embodiment,the linker is a peptide comprising one or more Phe residues, where atleast one Phe is about 7 to about 11, or about 7 to about 14 atoms fromthe binding ligand B. In another embodiment, the linker is a peptidecomprising Phe-Phe about 7 to about 11, or about 7 to about 14 atomsfrom the binding ligand B. It is to be understood that in each of theforegoing embodiments and variations, one or more Phe residues may bereplaced with Tyr, or another substituted variation thereof.

In another illustrative embodiment, the binding agent is a urea of anamino dicarboxylic acid, such as aspartic acid, glutamic acid, and thelike, and another amino dicarboxylic acid, or an analog thereof, and thelinker includes one or more aryl or arylalkyl groups, each of which isoptionally substituted, attached to the backbone of the linker. Inanother embodiment, the linker is a peptide comprising one or more arylor arylalkyl groups, each of which is optionally substituted, attachedto the backbone of the linker about 7 to about 11 atoms from the bindingligand B. In another embodiment, the linker is a peptide comprising twoaryl or arylalkyl groups, each of which is optionally substituted,attached to the backbone of the linker, where one aryl or arylalkylgroup is about 7 to about 11, or about 7 to about 14 atoms from thebinding ligand B, and the other aryl or arylalkyl group is about 10 toabout 14, or about 10 to about 17 atoms from the binding ligand B.

As described herein, the conjugates are targeted to cells that expressor over-express PSMA, using a PSMA binding ligand. Once delivered, theconjugates bind to PSMA. It is understood that in certain embodimentsthe conjugates remain on the surface of the cell for a period of timesufficient for imaging and/or diagnosis. In other embodiments, theconjugates are internalized in the cell expressing or over-expressingPSMA by endogenous cellular mechanisms, such as endocytosis, forsubsequent imaging and/or diagnosis, or treatment. Once internalized,the conjugates may remain intact or be decomposed, degraded, orotherwise altered to allow the release of the agent forming theconjugate. It is appreciated that in imaging and/or diagnosticconfigurations, the agent may remain intact as the conjugate or bereleased once it has been internalized into the targeted cell. It isfurther appreciated that in therapeutic configurations, the agent isadvantageously released from the conjugate once it has been internalizedinto the targeted cell.

In one illustrative embodiment, the drug is an imaging agent. In anotherillustrative variation, the drug is a diagnostic agent. In anotherillustrative variation, the drug is an chemotherapeutic agent.

In one aspect, the imaging agent is a radioisotope covalently attachedto the linker. In another aspect, the imaging agent is a radioactiveisotope, such as a radioactive isotope of a metal, coordinated to achelating group. Illustrative radioactive metal isotopes includetechnetium, rhenium, gallium, gadolinium, indium, copper, and the like,including isotopes ¹¹¹In, ^(99m)Tc, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, and thelike. Additional illustrative examples of radionuclide imaging agentsare described in U.S. Pat. No. 7,128,893, the disclosure of which isincorporated herein by reference. Additional illustrative chelatinggroups are tripeptide or tetrapeptides, including but not limited totripeptides having the formula:

wherein R is independently selected in each instance H, alkyl,heteroalkyl, cycloalkyl, heterocyclyl, alkenyl, alkynyl, aryl,heteroaryl, arylalkyl, heteroarylalkyl, and the like, each of which isoptionally substituted. It is to be understood that one R includes aheteroatom, such as nitro, oxygen, or sulfur, and is the point ofattachment of linker L. Illustratively, the following chelating groupsare described:

where X is oxygen, nitrogen, or sulfur, and where X is attached tolinker L, and n is an integer from 1 to about 5.

In another aspect, the imaging agent is a fluorescent agent. Fluorescentagents include OREGON GREEN fluorescent agents, including but notlimited to OREGON GREEN 488, OREGON GREEN 514, and the like, ALEXAFLUORfluorescent agents, including but not limited to ALEXAFLUOR 488,ALEXAFLUOR 647, and the like, fluorescein, and related analogs, BODIPYfluorescent agents, including but not limited to BODIPY F1, BODIPY 505,and the like, rhodamine fluorescent agents, including but not limited totetramethylrhodamine, and the like, DYLIGHT fluorescent agents,including but not limited to DYLIGHT 680, DYLIGHT 800, and the like, CW800, TEXAS RED, phycoerythrin, and others. Illustrative fluorescentagent are shown in the following illustrative general structures:

where X is oxygen, nitrogen, or sulfur, and where X is attached tolinker L; Y is OR^(a), NR^(a) ₂, or NR^(a) ₃ ⁺; and Y′ is O, NR^(a), orNR^(a) ₂ ⁺; where each R is independently selected in each instance fromH, fluoro, sulfonic acid, sulfonate, and salts thereof, and the like;and R^(a) is hydrogen or alkyl.

where X is oxygen, nitrogen, or sulfur, and where X is attached tolinker L; and each R is independently selected in each instance from H,alkyl, heteroalkyl, and the like; and n is an integer from 0 to about 4.

In another aspect, the imaging agent is a PET imaging agent, or a FRETimaging agent. PET imaging agents ¹⁸F, ¹¹C, ⁶⁴Cu, ⁶⁵Cu, and the like.FRET imaging agents include ⁶⁴Cu, ⁶⁵Cu. and the like. It appreciatedthat in the case of ¹⁸F, ¹¹C, the imaging isotope may be present on anypart of the linker, or alternatively may be present on a structureattached to the linker. For example in the case of ¹⁸F, fluoroarylgroups, such as fluorophenyl, difluorophenyl, fluoronitrophenyl, and thelike are described. For example in the case of ¹¹C, alkyl and alkyl arylare described.

In another aspect, the chemotherapeutic agent is a cytotoxic compound.The cytotoxic compounds described herein operate by any of a largenumber of mechanisms of action. Generally, cytotoxic compounds disruptcellular mechanisms that are important for cell survival and/or cellproliferation and/or cause apoptosis.

The drug can be any molecule capable of modulating or otherwisemodifying cell function, including pharmaceutically active compounds.Suitable molecules can include, but are not limited to: peptides,oligopeptides, retro-inverso oligopeptides, proteins, protein analogs inwhich at least one non-peptide linkage replaces a peptide linkage,apoproteins, glycoproteins, enzymes, coenzymes, enzyme inhibitors, aminoacids and their derivatives, receptors and other membrane proteins;antigens and antibodies thereto; haptens and antibodies thereto;hormones, lipids, phospholipids, liposomes; toxins; antibiotics;analgesics; bronchodilators; beta-blockers; antimicrobial agents;antihypertensive agents; cardiovascular agents includingantiarrhythmics, cardiac glycosides, antianginals and vasodilators;central nervous system agents including stimulants, psychotropics,antimanics, and depressants; antiviral agents; antihistamines; cancerdrugs including chemotherapeutic agents; tranquilizers;anti-depressants; H-2 antagonists; anticonvulsants; antinauseants;prostaglandins and prostaglandin analogs; muscle relaxants;anti-inflammatory substances; stimulants; decongestants; antiemetics;diuretics; antispasmodics; antiasthmatics; anti-Parkinson agents;expectorants; cough suppressants; mucolytics; and mineral andnutritional additives.

Further, the drug can be any drug known in the art which is cytotoxic,enhances tumor permeability, inhibits tumor cell proliferation, promotesapoptosis, decreases anti-apoptotic activity in target cells, is used totreat diseases caused by infectious agents, enhances an endogenousimmune response directed to the pathogenic cells, or is useful fortreating a disease state caused by any type of pathogenic cell. Drugssuitable for use in accordance with this invention includeadrenocorticoids and corticosteroids, alkylating agents, antiandrogens,antiestrogens, androgens, aclamycin and aclamycin derivatives,estrogens, antimetabolites such as cytosine arabinoside, purine analogs,pyrimidine analogs, and methotrexate, busulfan, carboplatin,chlorambucil, cisplatin and other platinum compounds, taxanes, such astamoxiphen, taxol, paclitaxel, paclitaxel derivatives, Taxotere®, andthe like, maytansines and analogs and derivatives thereof,cyclophosphamide, daunomycin, doxorubicin, rhizoxin, T2 toxin, plantalkaloids, prednisone, hydroxyurea, teniposide, mitomycins,discodermolides, microtubule inhibitors, epothilones, tubulysin,cyclopropyl benz[e]indolone, seco-cyclopropyl benz[e]indolone,O—Ac-seco-cyclopropyl benz[e]indolone, bleomycin and any otherantibiotic, nitrogen mustards, nitrosureas, vincristine, vinblastine,and analogs and derivative thereof such as deacetylvinblastinemonohydrazide, colchicine, colchicine derivatives, allocolchicine,thiocolchicine, trityl cysteine, Halicondrin B, dolastatins such asdolastatin 10, amanitins such as α-amanitin, camptothecin, irinotecan,and other camptothecin derivatives thereof, geldanamycin andgeldanamycin derivatives, estramustine, nocodazole, MAP4, colcemid,inflammatory and proinflammatory agents, peptide and peptidomimeticsignal transduction inhibitors, and any other art-recognized drug ortoxin. Other drugs that can be used in accordance with the inventioninclude penicillins, cephalosporins, vancomycin, erythromycin,clindamycin, rifampin, chloramphenicol, aminoglycoside antibiotics,gentamicin, amphotericin B, acyclovir, trifluridine, ganciclovir,zidovudine, amantadine, ribavirin, and any other art-recognizedantimicrobial compound.

Illustrative drugs and other therapeutic agents are described in U.S.Patent Application Publication Nos. US-2005-0002942-A1,US-2001-0031252-A1, and US-2003-0086900-A1. Illustrative imaging agentsand diagnostic agents are described in U.S. Patent ApplicationPublication No. US-2004-0033195-A1 and International Patent ApplicationPublication No. WO 03/097647. The disclosures of each of the foregoingpatent application publications are incorporated herein by reference.

The invention described herein also includes pharmaceutical compositionscomprising an amount of a binding ligand (B) drug delivery conjugateeffective to eliminate a population of pathogenic cells in a host animalwhen administered in one or more doses. The binding ligand drug deliveryconjugate is preferably administered to the host animal parenterally,e.g., intradermally, subcutaneously, intramuscularly, intraperitoneally,intravenously, or intrathecally. Alternatively, the binding ligand drugdelivery conjugate can be administered to the host animal by othermedically useful processes, such as orally, and any effective dose andsuitable therapeutic dosage form, including prolonged release dosageforms, can be used.

Examples of parenteral dosage forms include aqueous solutions of theactive agent, in an isotonic saline, 5% glucose or other well-knownpharmaceutically acceptable liquid carriers such as liquid alcohols,glycols, esters, and amides. The parenteral dosage form in accordancewith this invention can be in the form of a reconstitutable lyophilizatecomprising the dose of the drug delivery conjugate. In one aspect of thepresent embodiment, any of a number of prolonged release dosage formsknown in the art can be administered such as, for example, thebiodegradable carbohydrate matrices described in U.S. Pat. Nos.4,713,249; 5,266,333; and 5,417,982, the disclosures of which areincorporated herein by reference, or, alternatively, a slow pump (e.g.,an osmotic pump) can be used.

In one illustrative aspect, at least one additional compositioncomprising a therapeutic factor can be administered to the host incombination or as an adjuvant to the above-detailed methodology, toenhance the binding ligand drug delivery conjugate-mediated eliminationof the population of pathogenic cells, or more than one additionaltherapeutic factor can be administered. The therapeutic factor can beselected from a chemotherapeutic agent, or another therapeutic factorcapable of complementing the efficacy of the administered binding liganddrug delivery conjugate.

In one illustrative aspect, therapeutically effective combinations ofthese factors can be used. In one embodiment, for example,therapeutically effective amounts of the therapeutic factor, forexample, in amounts ranging from about 0.1 MIU/m²/dose/day to about 15MIU/m²/dose/day in a multiple dose daily regimen, or for example, inamounts ranging from about 0.1 MIU/m²/dose/day to about 7.5MIU/m²/dose/day in a multiple dose daily regimen, can be used along withthe binding ligand drug delivery conjugates to eliminate, reduce, orneutralize pathogenic cells in a host animal harboring the pathogeniccells (MIU=million international units; m²=approximate body surface areaof an average human).

In another embodiment, chemotherapeutic agents, which are, for example,cytotoxic themselves or can work to enhance tumor permeability, are alsosuitable for use in the method of the invention in combination with thebinding ligand drug delivery conjugates. Such chemotherapeutic agentsinclude adrenocorticoids and corticosteroids, alkylating agents,antiandrogens, antiestrogens, androgens, aclamycin and aclamycinderivatives, estrogens, antimetabolites such as cytosine arabinoside,purine analogs, pyrimidine analogs, and methotrexate, busulfan,carboplatin, chlorambucil, cisplatin and other platinum compounds,tamoxiphen, taxol, paclitaxel, paclitaxel derivatives, Taxotere®,cyclophosphamide, daunomycin, doxorubicin, rhizoxin, T2 toxin, plantalkaloids, prednisone, hydroxyurea, teniposide, mitomycins,discodermolides, microtubule inhibitors, epothilones, tubulysin,cyclopropyl benz[e]indolone, seco-cyclopropyl benz[e]indolone,O—Ac-seco-cyclopropyl benz[e]indolone, bleomycin and any otherantibiotic, nitrogen mustards, nitrosureas, vincristine, vinblastine,and analogs and derivative thereof such as deacetylvinblastinemonohydrazide, colchicine, colchicine derivatives, allocolchicine,thiocolchicine, trityl cysteine, Halicondrin B, dolastatins such asdolastatin 10, amanitins such as α-amanitin, camptothecin, irinotecan,and other camptothecin derivatives thereof, geldanamycin andgeldanamycin derivatives, estramustine, nocodazole, MAP4, colcemid,inflammatory and proinflammatory agents, peptide and peptidomimeticsignal transduction inhibitors, and any other art-recognized drug ortoxin. Other drugs that can be used in accordance with the inventioninclude penicillins, cephalosporins, vancomycin, erythromycin,clindamycin, rifampin, chloramphenicol, aminoglycoside antibiotics,gentamicin, amphotericin B, acyclovir, trifluridine, ganciclovir,zidovudine, amantadine, ribavirin, maytansines and analogs andderivatives thereof, gemcitabine, and any other art-recognizedantimicrobial compound.

The therapeutic factor can be administered to the host animal prior to,after, or at the same time as the binding ligand drug deliveryconjugates and the therapeutic factor can be administered as part of thesame composition containing the binding ligand drug delivery conjugateor as part of a different composition than the binding ligand drugdelivery conjugate. Any such therapeutic composition containing thetherapeutic factor at a therapeutically effective dose can be used inthe present invention.

Additionally, more than one type of binding ligand drug deliveryconjugate can be used. Illustratively, for example, the host animal canbe treated with conjugates with different vitamins, but the same drug ina co-dosing protocol. In other embodiments, the host animal can betreated with conjugates comprising the same binding ligand linked todifferent drugs, or various binding ligands linked to various drugs. Inanother illustrative embodiment, binding ligand drug delivery conjugateswith the same or different vitamins, and the same or different drugscomprising multiple vitamins and multiple drugs as part of the same drugdelivery conjugate could be used.

In another illustrative aspect, any effective regimen for administeringthe binding ligand drug delivery conjugates can be used. For example,the binding ligand drug delivery conjugates can be administered assingle doses, or can be divided and administered as a multiple-dosedaily regimen. In other embodiments, a staggered regimen, for example,one to three days per week can be used as an alternative to dailytreatment, and for the purpose of defining this invention suchintermittent or staggered daily regimen is considered to be equivalentto every day treatment and within the scope of this invention. In oneembodiment, the host is treated with multiple injections of the bindingligand drug delivery conjugate to eliminate the population of pathogeniccells. In another embodiment, the host is injected multiple times(preferably about 2 up to about 50 times) with the binding ligand drugdelivery conjugate, for example, at 12-72 hour intervals or at 48-72hour intervals. In other embodiments, additional injections of thebinding ligand drug delivery conjugate can be administered to thepatient at an interval of days or months after the initial injections(s)and the additional injections prevent recurrence of the disease statecaused by the pathogenic cells.

Illustratively, the binding ligand drug delivery conjugates can beadministered parenterally to the animal or patient suffering from thedisease state, for example, intradermally, subcutaneously,intramuscularly, intraperitoneally, or intravenously in combination witha pharmaceutically acceptable carrier. In another embodiment, thebinding ligand drug delivery conjugates can be administered to theanimal or patient by other medically useful procedures and effectivedoses can be administered in standard or prolonged release dosage forms.In another aspect, the therapeutic method can be used alone or incombination with other therapeutic methods recognized for treatment ofdisease states mediated by activated macrophages.

Described herein is a method for imaging pathogenic cell populationsthat express or over-express PSMA.

Described herein is a method for diagnosing diseases and disease statesthat are related to pathogenic cell populations that express orover-express PSMA. The compounds described herein bind selectivelyand/or specifically to cells that express or over-express PSMA. Inaddition, they not only show selectivity between pathogenic cells andnormal tissues, they show selectivity among pathogenic cell populations(see FIG. 8 where PSMA expressing LnCAP cells are preferentiallyvisualized compared to A549 tumors or KB tumors, which are not). Inaddition, the response is specific to PSMA binding as indicated bycompetition studies conducted with the conjugates described herein wherebinding is determined with the conjugate alone or in the presence ofexcess PMPA, a known binding ligand of PSMA. Binding at both the kidneyand tumor is blocked in the presence of excess PMPA (see, for example,Method Examples described herein).

In another embodiment, the conjugate has a binding constant K_(d) ofabout 100 nM or less. In another aspect, the conjugate has a bindingconstant K_(d) of about 75 nM or less. In another aspect, the conjugatehas a binding constant K_(d) of about 50 nM or less. In another aspect,the conjugate has a binding constant K_(d) of about 25 nM or less.

In another embodiment, the conjugates described herein exhibitselectivity for PSMA expressing or PSMA over-expressing cells or tissuesrelative to normal tissues such as blood, hear, lung, liver, spleen,duodenum, skin, muscle, bladder, and prostate, with at least 3-foldselectivity, or at least 5-fold selectivity. In one variation, theconjugates described herein exhibit selectivity for PSMA expressing orPSMA over-expressing cells or tissues relative to normal tissues with atleast 10-fold selectivity. It is appreciated that the selectivityobserved for imaging is indicative of the selectivity that may beobserved in treating disease states responsive to the selective orspecific elimination of cells or cell populations that express orover-express PSMA.

The unitary daily dosage of the drug delivery conjugate can varysignificantly depending on the host condition, the disease state beingtreated, the molecular weight of the conjugate, its route ofadministration and tissue distribution, and the possibility of co-usageof other therapeutic treatments such as radiation therapy. The effectiveamount to be administered to a patient is based on body surface area,patient weight, and physician assessment of patient condition. Effectivedoses can range, for example, from about 1 ng/kg to about 1 mg/kg, fromabout 1 μg/kg to about 500 μg/kg, from about 1 μg/kg to about 100 μg/kg,and from about 1 μg/kg to about 10 μg/kg.

Generally, any manner of forming a conjugate between the bivalent linker(L) and the binding ligand (B), or analog or derivative thereof, betweenthe bivalent linker (L) and the drug, or analog or derivative thereof,including any intervening heteroatoms, can be utilized in accordancewith the present invention. Also, any art-recognized method of forming aconjugate between the spacer linker, the releasable linker, and one ormore heteroatoms to form the bivalent linker (L) can be used. Theconjugate can be formed by direct conjugation of any of these molecules,for example, through hydrogen, ionic, or covalent bonds. Covalentbonding can occur, for example, through the formation of amide, ester,disulfide, or imino bonds between acid, aldehyde, hydroxy, amino,sulfhydryl, or hydrazo groups.

The synthetic methods are chosen depending upon the selection of theoptionally included heteroatoms or the heteroatoms that are alreadypresent on the spacer linkers, releasable linkers, the drug, and/or thebinding ligand. In general, the relevant bond forming reactions aredescribed in Richard C. Larock, “Comprehensive Organic Transformations,a guide to functional group preparations,” VCH Publishers, Inc. New York(1989), and in Theodora E. Greene & Peter G. M. Wuts, “Protective Groupsion Organic Synthesis,” 2d edition, John Wiley & Sons, Inc. New York(1991), the disclosures of which are incorporated herein by reference.

More specifically, disulfide groups can be generally formed by reactingan alkyl or aryl sulfonylthioalkyl derivative, or the correspondingheteroaryldithioalkyl derivative such as a pyridin-2-yldithioalkylderivative, and the like, with an alkylenethiol derivative. For example,the required alkyl or aryl sulfonylthioalkyl derivative may be preparedaccording to the method of Ranasinghe and Fuchs, Synth. Commun 18(3),227-32 (1988), the disclosure of which is incorporated herein byreference. Other methods of preparing unsymmetrical dialkyl disulfidesare based on a transthiolation of unsymmetrical heteroaryl-alkyldisulfides, such as 2-thiopyridinyl, 3-nitro-2-thiopyridinyl, and likedisulfides, with alkyl thiol, as described in WO 88/01622, EuropeanPatent Application No. 0116208A1, and U.S. Pat. No. 4,691,024, thedisclosures of which are incorporated herein by reference. Further,carbonates, thiocarbonates, and carbamates can generally be formed byreacting an hydroxy-substituted compound, a thio-substituted compound,or an amine-substituted compound, respectively, with an activatedalkoxycarbonyl derivative having a suitable leaving group.

EXAMPLES

The compounds described herein may be prepared by conventional organicsynthetic methods. In addition, the compounds described herein may beprepared as indicated below. Unless otherwise indicated, all startingmaterials and reagents are available from commercial supplies. All aminoacid starting materials were purchased from Chem-Impex Int (Chicago,Ill.). ¹H NMR spectra were obtained using a Bruker 500 MHz cryoprobe,unless otherwise indicated.

Example 1A

General synthesis of PSMA inhibitor intermediates for conjugation.Illustrated for specific synthesis of DUPA derivative2-[3-(1,3-Bis-tert-butoxycarbonyl-propyl)-ureido]-pentanedioic acid1-tert-butyl ester (I).

SK09. To a mixture of L-glutamate di-tert-butylester HCl (1.0 g, 3.39mmol) and triphosgene (329.8 mg, 1.12 mmol) in CH₂Cl₂ (25.0 mL) cooledto −78° C., triethylamine (1.0 mL, 8.19 mmol) was added. After stirringfor 2 h at −78° C. under nitrogen, mixture of L-Glu(OBn)-O-tert-Bu (1.2g, 3.72 mmol) and triethylamine (600 μL, 4.91 mmol) in CH₂Cl₂ (5.0 mL)was added. The reaction mixture was allowed to come to room temperatureover a period of 1 h and continued to stir at room temperatureovernight. The reaction mixture was washed with 1N HCl, brine and driedover Na₂SO₄. The crude product was purified using a flash chromatography(hexane:EtOAc=1:1, R_(t)=0.67) to give SK09 (1.76 g, 90.2%). C₃₀H₄₆N₂O₉;MW=578.69 g/mol; colorless oil; ¹H NMR (CDCl₃) δ 1.43 (s, 9H,CH₃-^(t)Bu); 1.44 (s, 9H, CH₃-^(t)Bu); 1.46 (s, 9H, CH₃-^(t)Bu); 1.85(m, 1H, Glu-H); 1.87 (m, 1H, Glu-H); 2.06 (m, 1H, Glu-H); 2.07 (m, 1H,Glu-H); 2.30 (m, 2H, Glu-H); 2.44 (m, 2H, Glu-H); 4.34 [s (broad), 1H,αH]; 4.38 [s (broad), 1H, α-H]; 5.10 (s, 2H, CH₂—Ar); 5.22 [s (broad),2H, Urea-H); 7.34 (m, 5H, Ar—H). ¹³C NMR (CDCl₃) δ 28.16; 28.25; 28.54;28.60; 30.52; 31.73; 53.13; 53.22; 66.58; 80.71; 82.25; 82.35; 128.39;128.71; 136.03; 156.96; 172.01; 172.16; 172.65; 173.13: CI-MS=579(M+H)⁺, ESI-MS=579 (M+H)⁺, 601 (M+Na adduct).

SK23. To a solution of compound SK09 (250 mg, 432 mmol) in CH₂Cl₂, 30%Pd/C (50 mg) was added. The reaction mixture was hydrogenated at 1 atm,room temperature for 24 h. Pd/C was filtered through celite pad andwashed with CH₂Cl₂. The crude product was purified using a flashchromatography (hexane:EtOAc=40:60, R_(t)=0.58) to give SK23 (169 mg,80.2%). C₂₃H₄₀N₂O₉; MW=488.57 g/mol; colorless oil; ¹H NMR (CDCl₃) δ1.46 (m, 27H, CH₃-^(t)Bu); 1.91 (m, 2H, Glu-H); 2.07 (m, 1H, Glu-H);2.18 (m, 1H, Glu-H); 2.33 (m, 2H, Glu-H); 2.46 (m, 2H, Glu-H); 4.31 (s(broad), 1H, αH); 4.35 (s (broad), 1H, α-H); 5.05 (t, 2H, Urea-H);CI-MS=489 (M+H)⁺, ESI-MS=489 (M+H)⁺, 511 (M+Na adduct), 487 (M−H)⁻.

Example 1B

General synthesis of PSMA inhibitor intermediates for conjugation.Illustrated for specific synthesis of tertiary butyl protected MUPAderivative2-[3-(1-tert-Butoxycarbonyl-2-mercapto-ethyl)-ureido]-pentanedioic aciddi-tert-butyl ester (II).

SKIS. To a mixture of L-glutamate di-tert-butylester HCl (200 mg, 0.676mmol) and triphosgene (67 mg, 0.228 mmol) in CH₂Cl₂ (5.0 mL), cooled to−78° C., triethylamine (50 μL, 0.410 mmol) was added. After stirring for2 h at −78° C. under nitrogen, mixture of D-Cys(Fm)-O^(t)Bu (291.4 mg,0.774 mmol) and triethylamine (30 μL, 240 mmol) in CH₂Cl₂ (1.0 mL) wasadded. The reaction mixture was allowed to come to room temperature overa period of 1 h and continued to stir at room temperature overnight. Thereaction mixture was washed with 1N HCl, brine and dried over Na₂SO₄.The crude product was purified using a flash chromatography(hexane:EtOAc=50:50, R_(t)=0.6) to give SK15 (374 mg, 86.4%).C₃₅H₄₈N₂O₇S; MW=640.83 g/mol; pale yellow oil; ¹H NMR (CDCl₃) δ 1.45 (s,27H, CH₃-^(t)Bu); 1.88 (m, 1H, Glu-H); 2.10 (m, 1H, Glu-H); 2.32 (m, 2H,Glu-H); 2.97 (m, 2H, Fm-CH2); 3.13 (m, 2H, Cys-H); 4.09 (t, 1H, Fm-H);4.38 (m, 1H, αH); 4.66 (m, 1H, α-H); 5.55 (d, 1H, Urea-H); 5.67 (d, 1H,Urea-H); 7.30 (q, 2H, Ar—H); 7.36 (q, 2H, Ar—H); 7.73 (m, 4H, Ar—H). ¹³CNMR (CDCl₃) δ 28.05; 28.14; 28.42; 31.64; 36.27; 37.25; 53.07; 53.73;80.51; 81.98; 82.42; 119.85; 124.95; 125.09; 127.09; 127.51; 141.09;145.99; 156.76; 170.80; 172.15; 172.43; CI-MS=641 (M+H)⁺, ESI-MS=641(M+H)⁺.

Example 2A

General synthesis of PSMA imaging agent conjugates. Illustrated bysynthesis of 14-atom linker compound SK28.

SK28 was synthesized using standard Fluorenylmethyloxycarbonyl (Fmoc)solid phase peptide synthesis (SPPS) starting from Fmoc-Cys(Trt)-Wangresin (Novabiochem; Catalog #04-12-2050). SK28 was purified usingreverse phase preparative HPLC (Waters, xTerra C₁₈ 10 μm; 19×250 mm)A=0.1 TFA, B=Acetonitrile (ACN); λ=257 nm; Solvent gradient: 5% B to 80%B in 25 min, 80% B wash 30 min run, (61%). Purified compounds wereanalyzed using reverse phase analytical HPLC (Waters, X-Bridge C₁₈ 5 μm;3.0×15 mm); A=0.1 TFA, B=ACN; λ=257 nm, 5% B to 80% B in 10 min, 80% Bwash 15 min run. C₄₇H₆₅N₂O₁₇S; MW=1060.13 g/mol; white solid; R_(t)=7.7min; ¹H NMR (DMSO-d₆/D₂O) δ 0.93 (m, 2H); 1.08 (m, 5H); 1.27 (m, 5H);1.69 (m, 2H); 1.90 (m, 2H); 1.94 (m, 2H); 2.10 (m, 2H); 2.24 (q, 2H);2.62 (m, 2H); 2.78 (m, 4H); 2.88 (dd, 1H); 2.96 (t, 2H); 3.01 (dd, 1H);3.31 (dd, 1H); 3.62 (dd, 1H); 3.80 (q, 1H, αH); 4.07 (m, 1H, αH); 4.37(m, 1H, αH); 4.42 (m, 2H, αH); 4.66 (m, 1H, αH); 7.18 (m, 10H, Ar—H):LC-MS=1061 (M+H)₊; ESI-MS=1061 (M+H)⁺.

Example 2AA

The following example compound was synthesized by an analogous process.

Examples 2B-2E

The following compounds were synthesized according to the processesdescribed herein using Fmoc SPPS starting from Fmoc-Cys(Trt)-Wang resin(Novabiochem; Catalog #04-12-2050), and purified using reverse phasepreparative HPLC (Waters, xTerra C₁₈ 10 μm; 19×250 mm) and analyzedusing reverse phase analytical HPLC (Waters, X-Bridge C₁₈ 5 μm; 3.0×15mm):

SK60 (0-atom linker): solvent gradient A=0.1 TFA, B=ACN; λ=220 nm;Solvent gradient: 1% B to 50% B in 25 min, 80% B wash 30 min run,(75.3%). C₂₁H₃₂N₆O₁₄S; MW=624.58 g/mol; white solid; R_(t)=6.3 min; ¹HNMR (DMSO-d₆/D₂O) δ 1.70 (m, 2H); 1.92 (m, 2H); 2.17 (m, 2H); 2.23 (m,2H); 2.57 (m, 1H); 2.77 (m, 4H); 3.45 (dd, 1H); 3.54 (dd, 1H); 3.83 (t,1H, αH); 4.06 (m, 1H, αH); 4.38 (m, 1H, α-H); 4.63 (m, 1H, α-H);ESI-MS=625 (M+H)⁺

SK62 (7 atom linker): solvent gradient A=0.1 TFA, TFA, B=ACN; λ=220, 257nm; Solvent gradient: 1% B to 50% B in 25 min, 80% B wash 30 min run,(72%). C₃₅H₄₈N₈O₁₈S; MW=900.86 g/mol; white solid; R_(t)=8.2 min; ¹H NMR(DMOS-d₆/D₂O) δ 1.62 (m, 1H); 1.70 (m, 2H); 1.79 (m, 1H); 1.90 (m, 2H);2.09 (t, 2H); 2.16 (m, 2H); 2.24 (m, 2H); 2.60 (m, 1H); 2.75 (m, 4H);2.81 (m, 1H); 2.97 (m, 1H); 3.33 (dd, 1H); 3.60 (dd, 1H); 3.81 (t, 1H,αH); 4.07 (m, 2H, αH); 4.33 [m, 1H, α-H]; 4.39 (t, α-H); 4.65 (m, 1H,α-H); 7.20 (m, 5H, Ar—H); ESI-MS=901 (M+H)⁺.

SK38 (16 atom linker): solvent gradient A=10 mM NH₄OAc, B=ACN; λ=257 nm;Solvent gradient: 1% B to 80% B in 25 min, 80% B wash 30 min run, (63%).C₄₃H₆₃N₉O₁₉S, MW=1042.07 g/mol; white solid; R_(t)=min; ¹H NMR(DMSO-d₆/D₂O) δ 0.94 (m, 2H); 1.08 (m, 5H); 1.27 (m, 5H); 1.66 (m, 2H);1.70 (m, 2H); 1.79 (m, 1H); 1.90 (m, 2H); 2.09 (t, 2H); 2.74 (m, 2H);2.84 (m, 1H); 2.95 (t, 3H); 3.07 (d, 2H); 3.23 (m, 1H); 3.43 (dd, 1H);3.52 (dt, 1H); 3.78 (m, 1H, αH); 3.81 (m, 1H, αH); 3.88 (m, 1H, αH);4.11 (m, 1H, αH); 4.39 [m, 2H, α-H]; 4.65 (m, 1H, α-H); 7.14 (m, 1H,Ar—H); 7.21 (m, 4H, Ar—H): ESI-MS=1043 (M+H)⁺.

SK57 (24 atom linker): solvent gradient A=0.1 TFA, B=ACN; λ=257 nm;Solvent gradient: 1% B to 50% B in 25 min, 80% B wash 30 min run, (56%).C₄₅H₇₀N₈O₂₂S, MW=1107.14 g/mol; colorless solid; ¹H NMR (DMSO-d₆/D₂O) δ1.66 (m, 2H); 2.07 (m, 4H); 2.31 (t, 1H); 2.43 (m, 1H); 2.77 (m, 2H);2.98 (dd, 1H); 3.14 (t, 2H); 3.24 (d, 1H); 3.40 (m, 4H, PEG-H); 3.46 (s,24H, PEG-H); 3.78 (t, 1H); 3.81 (t, 1H); 4.03 (m, 1H, αH); 4.40 (m, 2H,α-H); 7.16 (m, 1H, Ar—H); 7.22 (m, 4H, Ar—H): ESI-MS=1108 (M+H)⁺.

Example 2F

The following compound may be synthesized according to the processesdescribed herein.

Example 3A

General process for adding radionuclide to chelating group. Illustratedfor radio labeling of SK28 with ⁹⁹mTc to prepare SK33.

Preparation of SK28 formulation kits. HPLC grade Millipore filteredwater (50 mL) was added to a 100 mL bottle and argon was purged for atleast 10 min. Sodium α-D-glucoheptonate dihydrate (800 mg) was dissolvedin argon purged water (5 mL). Stannous chloride dihydrate (10 mg) wasdissolved in 0.02 M HCl (10 mL) while bubbling argon. Stannous chloride(0.8 mL) was added to the sodium glucoheptonate solution under argon.SK28 (1.4 mg) was added to the sodium glucoheptonate/stannous chloridesolution under argon. The pH of the reaction mixture was adjusted to6.8±0.2 using 0.1 N NaOH. Argon purged water (5.2 mL) was added to thereaction mixture to make total volume as 10 mL. 1.0 mL of reactionmixture was dispensed to each vial (10 vials) under argon atmosphere andlyophilized for 36-48 h. The vials were sealed with rubber stoppers andaluminum seals under argon atmosphere to make SK28 formulation kits. Theformulation kit vials were stored at −20° C. until they used.

Labeling SK28 with ⁹⁹mTc. Radio labeling of SK28 with ^(99m)Tc may beperformed according to published procedures. A formulation vial waswarmed to room temperature for 10 min and heated in a boiling water bathfor 3 min. Then 15 mCi of sodium pertechnetate ⁹⁹m Tc (1.0 mL) wasinjected and an equal volume of gas was withdrawn from the vial tonormalize the pressure. The vial was heated in the boiling water bathfor 15-20 min and then cooled to room temperature before using in theexperiment. Radiochemical purity was analyzed by radioactive TLC (>98%),that showed syn and anti isomers of the radio labeled compound(SK33/SK28-^(99m)Tc).

Examples 3B-3E

The following Examples were prepared according to the processesdescribed herein (both syn and anti isomers were obtained; only the synisomer is shown):

Example 3F

The following compound may be synthesized according to the processesdescribed herein.

Example 4

General synthesis of PSMA imaging agent conjugates illustrated for SK59using Universal PSMA (DUPA) resin, a 2-atom linker, and FITC. Thisconjugate may also be used for detecting circulating tumor cells inprostate cancer patients.

Synthesis of PSMA universal resin and SK59. Universal PSMA ligand (DUPA)resin was synthesized using Universal NovaTag™ resin (Novabiochem;Catalog #04-12-3910). Fmoc group was deprotected using 20%piperidine/DMF (N,N-dimethylformamide), after swelling the resin withDCM (CH₂Cl₂) and DMF. tert-Butyl protected DUPA was coupled using HATU[2-(1H-7-azabenzotriazol-1-yl)-,1,3,3-tetramethyl uroniumhexafluorophosphate] and DIPEA (N,N-diisopropylethylamine) in DMF. Thependant Mmt (4-Methoxytrityl) was removed with 1M HOBT(1-Hyroxybenzotriazole) in DCM/TFE (trifluoroethanol). The resinintermediate can be washed with DMF and used immediately in subsequentsynthetic steps or washed with DCM/DMF and then with MeOH, and dried forlater use.

Universal PSMA resin was reacted with commercially available FITC (1.25equiv) in the presence of DIPEA (4 equiv) in DMF to yield SK59 (2 atomlinker) construct. The final compound was cleaved from the resin using amixture of TFA (trifluoro acetic acid), TIPS (triisopropylsilane), andwater. Purification was by reverse phase preparative HPLC (Waters,xTerra C₁₈ 5 μm; 19×150 mm) A=10 mM NH₄OAc, B=ACN; δ=488 nm; Solventgradient: 1% B to 50% B in 25 m, 80% B wash 40 min run, (63%). SK59 wasanalyzed using reverse phase analytical HPLC (Waters, X-Bridge C₁₈ 5 μm;3.0×15 mm); A=10 mM NH₄OAc, B=ACN; λ=488 nm, 1% B to 50% B in 10 min,80% B wash 15 min run; C₃₄H₃₃N₆O₁₃S; MW=751.72 g/mol; orange colorsolid, R_(t)=7.2 min; ESI-MS=752 (M+H)⁺; 774 (M+Na)⁺; 750 (M−H)⁻.

Example 5A

General synthesis of PSMA imaging agent conjugates illustrated for SK64using Universal PSMA (DUPA) resin, a 16-atom linker, and FITC.

Universal PSMA resin was coupled with Fmoc-Glu-(O^(t)Bu)-OH andFmoc-EAOA (8-aminooctonoic acid) using standard Fmoc SPPS. Afterconjugating with fluoresceinisothiocyanate (1.25 equiv) in the presenceof DIPEA (4 equiv) in DMF, SK64 (16 atom linker) compound was cleavedfrom the resin using TFA/TIPS/H₂O. Purification was performed usingreverse phase preparative HPLC (Waters, xTerra C₁₈ 5 μm; 19×150 mm) A=10mM NH₄OAc, B=ACN; λ=488 nm; Solvent gradient: 1% B to 50% B in 25 min,80% B wash 40 min run, (57%). SK64 was analyzed using reverse phaseanalytical HPLC (Waters, X-Bridge C₁₈ 5 μm; 3.0×150 mm); A=10 mM NH₄OAc,B=ACN; λ=488 nm, 1% B to 50% B in 10 min, 80% B wash 15 min run;C₄₇H₅₅N₇O₁₇S; MW=1022.04 g/mol; orange color solid, R_(t)=7.8 min;ESI-MS=1022 (M+H)⁺; 1020 (M−H)⁻.

Examples 5B-5C

The following compounds were prepared using the synthetic processesdescribed herein:

SK63 (7-atom linker, C₃₉H₄₀N₆O₁₇, Mol. Wt.: 864.76). was prepared usinguniversal PSMA resin and standard Fmoc SPPS conjugated withFmoc-Glu-(O^(t)Bu)-OH. After coupling with FITC, compounds were cleavedfrom the resin using TFA/TIPS/H₂O cocktail and purified with reversephase preparative HPLC (Waters, xTerra C₁₈ 5 μm; 19×150 mm) A=10 mMNH₄OAc, B=ACN; λ=488 nm; Solvent gradient: 1% B to 50% B in 25 min, 80%B wash 40 min run, (65%); analyzed using reverse phase analytical HPLC(Waters, X-Bridge C₁₈ 5 μm; 3.0×150 mm); A=10 mM NH₄OAc, B=ACN; λ=488nm, 1% B to 50% B in 10 min, 80% B wash 15 min run; SK63: C₃₉H₄₀N₆O₁₆S;MW=880.83 g/mol; orange color solid, R_(t)=6.8 min; ESI-MS=881 (M+H)⁺;903 (M+Na)+; 863 (M−H)−.

SK58 (24-atom linker, C₄₉H₆₂N₆O₂₀S, Mol. Wt.: 1087.11) was preparedusing universal PSMA resin and standard Fmoc SPPS conjugated withFmoc-(PEG)₆-OH and purified by HPLC 1% B to 60% B in 25 min, 80% B wash40 min run, (65%); analyzed using reverse phase analytical HPLC (Waters,X-Bridge C₁₈ 5 μm; 3.0×150 min); A=10 mM NH₄OAc, B=ACN; λ=488 nm, 1% Bto 60% B in 10 min, 80% B wash 15 min run; C₄₉H₆₀N₆O₂₀S; MW=1087.11g/mol; orange color solid, R_(t)=7.3 min; ESI-MS=1087 (M+H)⁺; 1109(M+Na)+; 1085 (M−H)−.

Example 6A

General synthesis of Cys-maleimide PSMA imaging agent conjugatesillustrated for SK56 using Wang PSMA (DUPA) resin, a 28-atom linker, andOREGON GREEN 488, where n=3.

Related analogs where n is an integer from 4 to about 30 may also beprepared according to the processes described herein.

SK54 was prepared using standard Fmoc SPPS starting fromFmoc-Cys(Trt)-Wang resin (Novabiochem; Catalog #04-12-2050), purifiedusing reverse phase preparative HPLC (Waters, xTerra C₁₈ 10 μm; 19×250mm) A=0.1 TFA; B=ACN; λ=257 nm; Solvent gradient: 1% B to 60% B in 25min. 80% B wash 40 min run, (63%), and analyzed using reverse phaseanalytical HPLC (Waters, X-Bridge C₁₈ 5 μm; 3.0×50 mm); A=10 mM NH₄OAc,B=ACN; λ=257 nm, 1% B to 50% B in 10 min, 80% B wash 15 min run;C₃₈H₅₉N₅O₁₈S, MW=905.96 g/mol; colorless solid; R_(t)=9.2 min,LC-MS=906.3 g/mol; ESI-MS=906 (M+H)⁺; 904 (M−H)⁻.

SK56 (24 atom linker). HPLC grade Milli-Q water and satd NaHCO₃ werepurged with argon for 10 min. SK54 was dissolved in 1.0 mL of argonpurged water while bubbling argon. The pH of the solution was increasedup to 6.8 and OREGON GREEN 488 maleimide dissolved in 1.0 mL of THF wasadded to the reaction mixture. The reaction was monitored by analyticalHPLC (10 mM NH₄OAc, pH=7.0; 1% B to 50% B in 10 min 80% B wash 15 minrun) and reaction was completed within 10 min. THF was evaporated andreaction mixture was diluted with 5.0 mL of 7 mM phosphate buffer.Purification was performed using reverse phase preparative HPLC (Waters,xTerra C₁₈ 10 μm; 19×250 mm) A=7 mM Phosphate buffer pH=7.2, B=ACN;λ=488 nm; Solvent gradient: 1% B to 50% B in 25 min, 80% B wash 40 minrun, (89%); and analyzed using reverse phase analytical HPLC (Waters,X-Bridge C₁₈ 5 μm; 3.0×150 mm); A=10 mM NH₄OAc, B=ACN; λ=488 min, 1% Bto 50% B in 10 min, 80% B wash 15 min run; C₆₂H₇₀F₂N₆O₂₅S; MW=1369.31g/mol; orange color solid, R_(t)=7.0 min; LC-MS=1370.2; ESI-MS=1391(M+Na)⁺.

The following 24-atom linker compounds were prepared in an analogousmanner to those described herein using the General syntheses describedherein.

Example 6B

The following ALEXAFLUOR 488 conjugate compound was prepared accordingto the processes described herein starting with SK55, where n=3.

Related analogs where n is an integer from 4 to about 30 may also beprepared according to the processes described herein.

Examples 7A-7C

The following DUPA imaging agent conjugate compounds, SK51, SK45, andSK49 were prepared according to the processes described herein, where nis 5:

SK51 (25-atom linker, and ALEXAFLUOR 647, MW ˜2300 (commerciallyavailable from Invitrogen))Related analogs where n is an integer from 0 to about 12 may also beprepared according to the processes described herein.

SK45 (25 Atom linker BODIPY 505, C67H87BF2N13O20S, Mol. Wt.: 1475.35)Related analogs where n is an integer from 0 to about 12 may also beprepared according to the processes described herein.

SK49 (25 Atom linker—OREGON GREEN 488, C71H76F2N10O24, Mol. Wt.:1523.48) Related analogs where n is an integer from 0 to about 12 mayalso be prepared according to the processes described herein.

Synthesis of the Linker. In each of the foregoing Examples, the linkerwas synthesized using standard Fmoc SPPS starting fromFmoc-Cys(Trt)-Wang resin (Novabiochem; Catalog #04-12-2050);C₄₇H₆₅N₉O₁₇S; MW=1060.13 g/mol; white solid; R_(t)=7.7 min; ¹H NMR(DMSO-d₆/D₂O) δ 0.93 (m, 2H); 1.08 (m, 5H); 1.27 (m, 5H); 1.69 (m, 2H);1.90 (m, 2H); 1.94 (m, 2H); 2.10 (m, 2H); 2.24 (q, 2H); 2.62 (m, 2H);2.78 (m, 4H); 2.88 (dd, 1H); 2.96 (t, 2H); 3.01 (dd, 1H); 3.31 (dd, 1H);3.62 (dd, 1H); 3.80 (q, 1H, αH); 4.07 (m, 1H, αH); 4.37 (m, 1H, αH);4.42 (m, 2H, αH); 4.66 (m, 1H, αH); 7.18 (m, 10H, Ar—H): LC-MS=1061(M+H)₊; ESI-MS=1061 (M+H)₊.

Synthesis of SK51 (ALEXAFLUOR 647 conjugate), SK45 (BODIPY conjugate)and SK49 (OREGON GREEN 488 conjugate). HPLC grade Milli-Q water and satdNaHCO₃ were purged with argon for 10 min. Linker was dissolved in 1.0 mLof argon purged while bubbling argon.

The pH of the solution was increased to 6.8 and ALEXAFLUOR maleimide,BODIPY maleimide, or OREGON GREEN 488 maleimide, respectively, wasdissolved in 1.0 mL of tetrahydrofuran (THF) was added to the reactionmixture. Progress of the reaction was monitored by analytical HPLC (10mM NH₄OAc, pH=7.0; 1% B to 50% B in 10 min 80% B wash 15 min run) andreaction was completed within 10 min. THF was evaporated and reactionmixture was diluted with 5.0 mL of 1 mM phosphate buffer (pH=7.2).

Compounds were purified using reverse phase preparative HPLC (Waters,xTerra C₁₈ 5 μm; 18×150 mm) A=1 mM Phosphate buffer pH=7.2, B=ACN: λ=647or 488 nm: Solvent gradient: 1% B to 50% B in 25 min, 80% B wash 40 minrun; and analyzed using reverse phase analytical HPLC (Waters, X-BridgeC₁₈ 5 μm; 3.0×50 mm); A=10 mM NH₄OAc, B=ACN; λ=588 or 488 nm, 1% B to50% B in 10 min, 80% B wash 15 min run.

SK51: MW ˜2360.13 g/mol; blue color solid, R_(t)=6.7 min; (structure ofthe ALEXAFLUOR 647 is not known);

SK45: C₆₇H₈₇BF₂N₁₃O₂₀S; MW=1475.35 g/mol; orange color solid, R_(t)=7.6min; LC-MS=1475.3 (M+H)⁺;

SK49: C₇₁H₇₆F₂N₁₀O₂₄S; MW=1523.48 g/mol; orange color solid, R_(t)=6.7min; LC-MS=1524 (M+H)⁺.

Example 8A

General synthesis of PSMA disulfide linker intermediate for releasableagent conjugate, illustrated for SK68.

SK68 was synthesized using standard Fmoc SPPS starting fromFmoc-Cys(Trt)-Wang resin (Novabiochem; Catalog #04-12-2050), purifiedusing reverse phase preparative HPLC (Waters, xTerra C₁₈ 10 μm; 19×250mm) A=0.1 TFA, B=ACN; λ=257 nm; Solvent gradient: 1% B to 50% B in 30min. 80% B wash 40 min run, (68%); and analyzed using reverse phaseanalytical HPLC (Waters, X-Bridge C₁₈ 5 μm; 3.0×15 mm); A=0.1 TFA,B=ACN; λ=257 nm, 1% B to 50% B in 10 min. 80% B wash 15 ruin run.C₃₂H₄₂N₆O₁₇S; MW=814.77 g/mol; white solid; R_(t)=8.2 min; ¹H NMR(DMOS-d₆/D₂O) δ 1.70 (m, 3H); 1.90 (m, 3H); 2.10 (m, 2H); 2.17 (m, 2H);2.23 (m, 2H); 2.36 (m, 1H); 2.59 (dd, 1H); 2.79 (m, 3H); 3.04 (dd, 1H);4.07 (m, 2H, αH); 4.13 (m, 1H, αH); 4.37 [m, 1H, α-H]; 4.47 (m, 2H,α-H); 7.19 (m, 5H, Ar—H); 7.87 (d, Ures-NH); 8.20 (d, 1H, Urea-NH);LC-MS=815.3 (M+H)⁺.

Example 8B

General synthesis of PSMA disulfide linker intermediate for releasableagent conjugate, illustrated for SK28L.

SK28 was synthesized using standard Fmoc-SPPS starting fromFmoc-Cys(Trt)-Wang resin (Novabiochem; Catalog #04-12-2050); purifiedusing reverse phase preparative HPLC (Waters, xTerra C₁₈ 10 μm; 19×250mm) A=0.1 TFA, B=ACN; λ=257 nm; Solvent gradient: 5% B to 80% B in 25min, 80% B wash 30 min run, (61%); and analyzed using reverse phaseanalytical HPLC (Waters, X-Bridge C₁₈ 5 μm; 3.0×15 mm); A=0.1 TFA,B=ACN; λ=257 nm, 5% B to 80% B in 10 min. 80% B wash 15 min run. SK28L:C₄₇H₆₅N₂O₁₇S; MW=1060.13 g/mol; white solid; R_(t)=7.7 min; ¹H NMR(DMSO-d₆/D₂O) δ 0.93 (m, 2H); 1.08 (m, 5H); 1.27 (m, 5H); 1.69 (m, 2H);1.90 (m, 2H); 1.94 (m, 2H); 2.10 (m, 2H); 2.24 (q, 2H); 2.62 (m, 2H);2.78 (m, 4H); 2.88 (dd, 1H); 2.96 (t. 2H); 3.01 (dd, 1H); 3.31 (dd, 1H);3.62 (dd, 1H); 3.80 (q, 1H, αH); 4.07 (m, 1H, αH); 4.37 (m, 1H, αH);4.42 (m, 2H, αH); 4.66 (m, 1H, αH); 7.18 (m, 10H, Ar—H): LC-MS=1061(M+H)₊; ESI-MS=1061 (M+H)⁺.

Example 9A

General synthesis for preparing disulfide-linked conjugates, illustratedfor tubulysin B conjugate SK71 (20-atom linker).

Synthesis of EC0312. Tubulysin B (30 mg, 0.036 mmol) was dissolved inethylacetate (600 μL) under argon at −15° C. Isobutyl chlorofomate (4.7μL, 0.054 mmol) and diisopropylethylamine (13.2 μL. 0.076 mmol) wereadded to the reaction mixture; reaction was stirred at −15° C. for 45min under argon. EC0311 (13.4 mg, 0.054 mmol) dissolved in ethylacetate(500 μL) was added. Reaction mixture was stirred at −15° C. for another15 min and then at room temperature for 45 min. Solvent was evaporatedand residue was purified using short column (2%-8% methanol in CH₂Cl₂)to get EC0312 (34.4 mg, 90.5%). EC0312 was characterized using NMR(Varian 300 MHz, in CDCl₃), and LC-MS=1058.3 (M+H)⁺.

Synthesis of SK71. HPLC grade Milli-Q water and satd NaHCO₃ were purgedwith argon for 10 min. SK68 was dissolved in 1.0 mL of argon purgedwater while bubbling argon through the solution. The pH of the solutionwas increased to 6.8 using argon purged NaHCO₃ and EC0312 dissolved inTHF (2.0 mL) was added to the reaction mixture. Progress of the reactionwas monitored by analytical HPLC (10 mM NH₄OAc, pH=7.0; λ=254; 1% B to50% B in 10 min 80% B wash 15 min run) and reaction was completed within10 min. THF was evaporated and reaction mixture was diluted with 5.0 mLof 2 mM phosphate buffer. SK71 (61.3%) was purified using reverse phasepreparative HPLC (Waters, xTerra C₁₈ 10 μm; 19×250 mm) A=2 mM Phosphatebuffer, B=ACN; δ=254 nm; 5% B to 80% B in 25 min 80% B wash 40 min run;and analyzed using reverse phase analytical HPLC (Waters, X-Bridge C₁₈ 5μm; 3.0×15 mm); A=10 mM NH₄OAc, B=ACN; λ=254 nm, 1% B to 50% B in 10min, 80% B wash 15 min run. C₇₇H₁₀₉N₁₃O₂₈S₃: MW=1760.95 g/mol; whitecolor solid, R_(t)=7.6 min; ¹H NMR (DMSO-d₆/D₂O) was consistent with theSK71 structure; HRMS (MALDI) (m/z): (M−H)− calcd. for, C₇₇H₁₁₀N₁₃O₂₈S₃,1758.6594; found, 1758.7033; LRMS (LCMS) (m/z): (M+H)+ calcd. for1761.9; found, 1761.8; UV/Vis: λmax=254 nm.

Example 9B

Similarly, the D-Cys analog of SK71 was prepared as described herein.

Example 9C

General synthesis for preparing disulfide-linked conjugates, illustratedfor tubulysin B conjugate SK77 (31-atom linker).

HPLC grade Milli-Q water and satd NaHCO₃ were purged with argon for 10min SK68 was dissolved in 1.0 mL of argon purged water while bubblingargon. The pH of the solution was increased to 6.8 using argon purgedNaHCO₃ and EC0312 dissolved in THF (2.0 mL) was added to the reactionmixture. Progress of the reaction was monitored by analytical HPLC (10mM NH₄OAc, pH=7.0; λ=254; 1% B to 50% B in 10 min 80% B wash 15 min run)and reaction was completed within 10 min. THF was evaporated andreaction mixture was diluted with 5.0 mL of 2 mM phosphate buffer. SK77(61%) was purified using reverse phase preparative HPLC (Waters, xTerraC₁₈ 10 μm; 19×250 mm) A=2 mM phosphate buffer, B=ACN; λ=254 nm; 5% B to80% B in 25 min 80% B wash 40 min run; and analyzed using reverse phaseanalytical HPLC (Waters, X-Bridge C₁₈ 5 μm; 3.0×15 mm); A=10 mM NH₄OAc,B=ACN; λ=254 nm, 1% B to 50% B in 10 min, 80% B wash 15 min run.C₉₃H₁₃₃N₁₆O₉₈S₃: MW=2006.32 g/mol; white color solid, R_(t)=7.7 min; ¹HNMR (DMSO-d₆/D₂O); LC-MS=2007.0 (M+H)⁺.

Example 9D

Similarly, the D-Cys analog of SK77 was prepared as described herein.

Example 9E

Similarly, the D-Cys, propanoic acid analog of SK77 was prepared asdescribed herein.

Examples 9F-9G

The following DUPA vinblastine and DUPA camptothecin compounds, SK37 andSK45, respectively, were prepared according to the processes describedherein.

SK37 (vinblastine conjugate, C₉₃H₁₂₃N₁₅O₂₆S₇, Mol. Wt.: 1931.19)prepared in 63.1% yield. C₉₃H₁₂₃N₁₅O₂₆S₂: MW=1931.19 g/mol; white colorsolid, R_(t)=7.7 min; ¹H NMR (DMSO-d₆/D₂O); LC-MS=1932.6 (M+H)⁺.

SK45 (camptothecin conjugate, C₇₀H₈₃N₁₁O₂₃S₂, Mol. Wt.: 1510.60)prepared in 66% yield. C₇₀H₈₃N₁₁O₂₃S₂: MW=1510.60 g/mol; white colorsolid, R_(t)=7.5 min; ¹H NMR (DMSO-d₆/D₂O); LC-MS=1511.1 (M+H)⁺.

Example 9H

Similarly, the Glu-Asp-Phe analog of SK37 was prepared as describedherein.

Examples 10

The following compounds were prepared using the synthetic processesdescribed herein:

DOTA conjugate capable of chelating for example ⁶⁴Cu, ⁶⁵Cu, and the like

DOTA conjugate capable of chelating for example ⁶⁴Cu, ⁶⁵Cu, and the like

DTPA conjugate capable of chelating for example In, Ga, Ir, Yr, and thelike

Tripeptide conjugate capable of chelating for example Tc, Tc oxides, andthe like

The foregoing exemplary embodiments are intended to be illustrative ofthe invention, and should not be interpreted or construed as limiting inany way the invention as described herein.

METHOD EXAMPLES Example 1A

In Vitro Binding Studies Using Lncap Cells and SK28 (14 Atom Spacer).LNCaP cells (a human prostate cancer cell line over-expressing PSMA,purchased from American Type Culture Collection (ATCC)) were seeded intwo 24-well (120,000 cells/well) falcon plates and allowed to grow toadherent monolayers for 48 hours in RPMI with glutamine (2 mM)(GibcoRPMI medium 1640, catalog #22400) plus 10% FBS (Fetal Bovine Serum), 1%sodium pyruvate (100 mM) and 1% PS (penicillin streptomycin) in a 5%-CO2atmosphere at 37° C. Cells of one 24-well plate were incubated withincreasing concentrations of SK28-99mTc from 0 nM-450 nM (triplicatesfor each concentration) in a 5%-CO2 atmosphere at 37° C. for 1 hour.Cells of the second 24-well plate were incubated with 50 uM PMPA in a5%-CO2 atmosphere at 37° C. for 30 minutes, then incubated withincreasing concentrations of SK28-99mTc from 0 nM-450 nM (triplicatesfor each concentration) in a 5%-CO2 atmosphere at 37° C. for 1 hour(competition study). Cells were rinsed three times with 1.0 mL of RPMI.Cells were lysed with tris-buffer, transferred to individual gammascintigraphy vials, and radioactivity was counted. The plot of cellbound radioactivity verses concentration of radiolabeled compound wasused to calculate the Kd value. The competition study was used todetermine the binding specificity of the ligand (DUPA) to the PSMA (FIG.1A).

Example 1B

In Vitro Binding Studies Using LNCaP Cells and SK33 (14 atom spacer).LNCaP cells (150,000 cells/well) were seeded onto 24-well Falcon platesand allowed to form confluent monolayers over 48 h. Spent medium in eachwell was replaced with fresh medium (0.5 mL) containing increasingconcentrations of DUPA-99mTc in the presence (▴) or absence (▪) ofexcess PMPA. After incubating for 1 h at 37° C., cells were rinsed withculture medium (2×1.0 mL) and tris buffer (1×1.0 mL) to remove anyunbound radioactivity. After suspending cells in tris buffer (0.5 mL),cell bound radioactivity was counted using a γ-counter (Packard, PackardInstrument Company). The dissociation constant (KD) was calculated usinga plot of cell bound radioactivity versus the concentration of theradiotracer using nonlinear regression in GraphPad Prism 4. Error barsrepresent 1 standard deviation (n=3). Experiment was performed threetimes with similar results. (FIG. 1B).

Example 2

Quantification of PSMA Molecules on LNCaP Cells. LNCaP cells were seededin a 24-well falcon plate and allowed to grow to adherent monolayers for48 hours in RPMI (Gibco RPMI medium 1640, catalog #22400) plus 10% FBS(Fetal Bovine Serum), 1% glutaric and 1% PS (penicillin streptomycin) ina 5%-CO2 atmosphere at 37° C. Cells were then incubated with increasingconcentrations of SK28-99mTc from 0 nM-450 nM (triplicates for eachconcentration) in a 5%-CO2 atmosphere at 4° C. or at 37° C. for 1 hour.Cells were rinsed three times with 1.0 mL of RPMI. Cells were lysed withtris-buffer, transferred to individual gamma scintigraphy vials, andradioactivity was counted. The plot of cell bound radioactivity versesconcentration of radiolabeled compound was used to calculate number ofPSMA/LNCaP cell. The radioactivity of a 30 nM sample of SK28-99mTc (20uL) was counted. At 4° C. (to prevent endocytosis of PSMA), the numberof moles in the 30 nM sample=30 nM×20 uL=(30×10-9 mol/L)×(20×10-6L)=6×10-13 mol. The number of atoms in the 30 nM sample=(6×10-13mol)×(6.023×1023 atom/mol)=3.6×1011 atom. The radio count of 20 uL ofthe 30 nM sample=20477 cpm (cpm/atom=3.6×1011/20477=1.76×107). The cellbound radioactivity at the saturation point at 4° C.=12 000 cpm. Thenumber of atoms at the saturation point=(1.76×107 atom)×(12 000 cpm).The number of cells/well=245,000. The number of PSMA/cell at 4°C.=(2.12×1011)/2.45×105=864 396.4˜0.9×106 PSMA/LNCaP cell.

The cell bound radioactivity at the saturation point at 37° C.=33,000cpm (approximately three fold higher than at 4° C.). This shows thatPSMA undergoes endocytosis, unloading the drug and recycling, similar tocell surface receptors. See FIG. 2.

Example 3

Spacer-Dependent Binding Studies. LNCaP cells were seeded in 24-well(120,000 cells/plate) falcon plates (10 plates) and allowed to grow toadherent monolayers for 48 hours in RPMI (Gibco RPMI medium 1640,catalog #22400) plus 10% FBS (Fetal Bovine Serum), 1% sodium pyruvateand 1% PS (penicillin streptomycin) in a 5%-CO2 atmosphere at 37° C.Cells were then incubated with increasing concentrations of SK60-99mTc(zero atom spacer), SK62-99mTc (7 atom spacer), SK28-99mTc (14 atomspacer), SK38-99mTc (16 atom spacer) and SK57-99mTc (24 atom spacer)from 0 nM-1280 nM (triplicates for each concentration) in a 5%-CO2atmosphere at 37° C. for 1 hour. Also, in separate plates, cells wasincubated with 50 uM PMPA in a 5%-CO2 atmosphere at 37° C. for 30minutes and then incubated with increasing concentration of SK60-99mTc(zero atom spacer), SK62-99mTc (7 atom spacer), SK28-99mTc (14 atomspacer), SK38-99mTc (16 atom spacer) and SK57-99mTc (24 atom spacer)from 0 nM-1280 nM (triplicates for each concentration) in a 5%-CO2atmosphere at 37° C. for 1 hour (competition studies; data not shown).Cells were rinsed three times with 1.0 mL of RPMI. Cells were lysed withtris-buffer, transferred to individual gamma scintigraphy vials, andradioactivity was counted. The plot of cell bound radioactivity versesconcentration of the radiolabeled compound was used to calculate the Kdvalue. The plot of % saturation verses concentration of the radiolabeledcompound as well as the plot for Kd verses spacer length are shown(FIGS. 3A and B).

Example 4

In Vivo Growth of Human LNCaP Tumor Cells in Nude Mice. LNCaP cells weremaintained in RPMI 1640 (Gibco RPMI medium 1640, catalog #22400) withglutamine (2 mM), 10% FBS (Fetal Bovine Serum), 1% sodium pyruvate (100mM) and 1% PS (penicillin streptomycin) in a 5%-CO2 atmosphere at 37° C.Four to five week-old athymic male nude mice (nu/nu) were obtained fromthe NCI Charles River and maintained in a sterile environment. Mice werehoused in polycarbonate shoebox cages with wire top lids and maintainedon a normal diet. Mice were allowed to acclimate for one week prior toinoculation of LNCaP cells. Matrigel and high concentrated (HC) matrigelwere purchased from BD Biosciences. Nude mice were inoculated witheither 2.5×106 or 5.0×106 in vitro propagated LNCaP cells in 50%matrigel (100 uL RPMI medium+100 uL of matrigel) or 50% highconcentrated matrigel (100 uL RPMI medium+100 uL of HC matrigel) todetermine optimal conditions, including number of cells, vehicle, etc.Cells were subcutaneously injected into each axial and each flank of thenude mice to determine the optimal site. The volume of each tumor wasmeasured in perpendicular directions twice a week using a caliper andbody weight was measured once a week (data not shown).

The volume of each tumor was calculated as 0.5×L×W2, where L=measurementof longest axis in millimeters and W=measurement of axis perpendicularto L in millimeters. Approximately 5.0×106 LNCaP cells in 50% HCmatrigel on the axial gave 600 mm3 tumors within 30 days. See FIG. 4.

Example 5

Comparison of lncap, kb and a549 Cell Tumor Growth in Mice. LNCaP, KB,and A549 cells were maintained in RPMI 1640 (Gibco RPMI medium 1640,catalog #22400) with glutamine (2 mM), 10% FBS (Fetal Bovine Serum), 1%sodium pyruvate (100 mM) and 1% PS (penicillin streptomycin) in a 5%-CO2atmosphere at 37° C. Four-five weeks old male nude mice (nu/nu) wereobtained from the NCI Charles River and maintained in a sterileenvironment. Mice were housed in polycarbonate shoebox cages with wiretop lids and maintained on a normal diet. Mice were allowed to acclimatefor one week prior to inoculation of cells.

For tumor cell inoculation, 5.0×106 LNCaP cells in 50% high concentratedmatrigel, 1.0×106 KB cells in RPMI medium, or 1.0×10 6 A549 cells inRPMI medium were subcutaneously injected into the right axial (someanimals were injected in both) of the nude mice. The volume of eachtumor was measured in two perpendicular directions twice a week using acaliper (See FIGS. 5A and 5B) and body weight was measured once a week(data not shown). The volume of the tumors were calculated as 0.5×L×W2,where L=measurement of longest axis in millimeters and W=measurement ofaxis perpendicular to L in millimeters.

Example 6A

In Vivo Imaging of Tumors in Mouse Using PSMA-99mTc. When tumors reacheda volume of between 500-600 mm3, 99mTc-labeled compounds (e.g.SK28-99mTc, SK60-99mTc, etc) prepared as described, were administeredthrough intraperitoneal injection (subcutaneously). Four hours later,animals were euthanized and blood was taken by cardio punch andtransferred to individual gamma scintigraphy vials per each animal. Theimaging experiments were carried out using either a Kodak or gammascintigraphic camera imager (FIGS. 6A, 6B, 6C, 7A, 7B and 7C). [Note:PMPA was injected 30 minutes before injecting SK28-99mTc. Other thanuptake in the cancerous masses, SK28-99mTc distribution was limited tokidneys (FIGS. 6A, 6B and 6C). Both mice (FIGS. 7A, 7B and 7C) wereinjected with SK60-99mTc and distribution was limited mostly to thekidneys (no tumor uptake even after shielding both kidneys.)]

FIGS. 6A, 6B, and 6C show images of mice with human LNCaP tumors usingSK28-99mTc (radiolabeled 14 Atom spacer). FIGS. 6A-6C represent 3separate sets of mice: the left hand image shows white light images andthe right hand image shows an overlay of the radioimage with the whitelight image of mice with LNCaP tumors imaged using a Kodak camera imager4 hours after subcutaneous (administered through intraperitoneal)injection of 1 ng/kg SK28-99mTc without [left mouse in each set ofimages] and with 50 mg/kg PMPA [right mouse in both sets of images] toblock PSMA (as a competitor). FIG. 6D shows a single mouse study forLNCaP tumors imaged using Kodak imager 4 hours after subcutaneous(administered through intraperitoneal) injection of 1 ng/kg SK28-99mTcshowing in the left hand image an overlay of radioimage with kidneyshield and white light image with no shield and in the right hand imagean overlay of radioimage with kidney shield and X-ray image with noshield.

Example 6B

In Vivo Imaging of Tumors in Mouse Using dupa-99mTc. To furtherestablish the specificity of our DUPA conjugates for prostate cancercells, DUPA-99mTc was injected intraperitoneally (i.p.) into athymicnude mice bearing LNCaP tumors on their shoulders. After 4 h to allowfor clearance of unbound conjugate, the distribution of the retainedDUPA-99mTc was imaged by gamma scintigraphy. As seen in FIGS. 7D(a) and7D(c), the targeted 99mTc radiotracer accumulated mainly in the PSMApositive LNCaP tumor, with little or no radioactivity in other tissuesexcept the kidneys. Importantly, kidney uptake may be peculiar to themouse, since immunohistochemical and RT-PCR analyses suggest that PSMAexpression is high in murine kidneys but minimal in human kidneys. Invivo specificity of the PSMA-targeted imaging agent was further testedby prior administration of excess PMPA to block all PSMA sites beforeDUPA-99mTc administration. As shown in FIGS. 7D(b) and 7D(d), blockedLNCaP tumors display no DUPA-99mTc uptake, confirming the specificity ofthe DUPA conjugate for PSMA in vivo. To further document thisspecificity, the radiotracer was also administered to two PSMA negativemouse xenograft models [A549 (a human lung cancer cell line) and KB (ahuman nasopharyngeal cancer cell line)], and again whole body imageswere taken. As anticipated, no radioactivity was observed in either KBor A549 tumors (FIGS. 7D(e) and 7D(f)), even after shielding of thekidneys was performed to allow detection of low levels of DUPA-99mTc inother tissues. These studies thus confirm that very little DUPA-99mTcbinding occurs to sites unrelated to PSMA in vivo.

FIG. 7D shows the whole body images of solid tumor xenografts in nu/numice taken 4h after injection of 150 μCi DUPA-99mTc. Overlay ofwhole-body radioimages on white light images of mice bearing LNCaPtumors that were treated with DUPA-99mTc in the absence 7D(a, c) orpresence 7D(b, d) of 100-fold molar excess PMPA. Overlay of radioimageson white light images were also obtained of mice bearing an A549 tumor7D(e) or a KB tumor 7D(f) that were similarly treated with DUPA-99mTc.Except in images 7D(a) and 7D(b), kidneys were shielded with lead pads.All images were taken using a Kodak Imaging Station 4 h after anintraperitoneal injection of DUPA-99mTc. Arrows indicate solid tumorxenografts. Similar images were obtained on all 5 mice in each treatmentgroup.

Example 7A

Biodistribution Studies. After imaging, all animals were dissectedapproximately 6-7 h after administering SK28-99mTc or SK60-99mTc [orother radiolabeled compounds (data not shown)] and organs (blood, tumor,heart, liver, kidney, spleen, skin, muscle, etc) were transferred toindividual gamma scintigraphy vials for each animal and radioactivitywas counted. Note: blood samples were collected (using cardio punch)immediately after sacrificing the animal and before imaging the animal.The plot of tumor to tissue cpm/g ratio verses tissue was used todetermine bio-distribution of the imaging agent (FIGS. 8A and 8B).

Example 7B

Biodistribution studies of DUPA-99mTc in nu/nu mice bearing LNCaP, A549,or KB tumors. Tumor-bearing mice were euthanized 4 h afterintraperitoneal injection of DUPA-99mTc (50 μmol/kg, 150 μCi) andtissue-accumulated radioactivity was counted using a γ-counter. Thepercent injected dose per gram wet tissue was calculated as described inthe Methods. The data were obtained in a single experiment and errorbars represent s.d. (n=5). LNCaP tumors (solid bars), LNCaP tumors inmice pre-injected with 100-fold molar excess of PMPA (open bars), A549tumors (cross-hatched bars), KB tumors (horizontally-hatched bars) (FIG.8C).

Example 8

Single dose toxicity in live mice. Administration of SK71 was in asingle dose as indicated. The data show that the MTD for the conjugateis about 4.5 μmol/kg for single dosing. (See FIG. 9A)

Example 9

Multiple dose toxicity in live mice. SK71 was administered in 5 doses onalternate days (M, W, F, M, W). The data show that the MTD for SK71 is 2μmol/kg for multiple dosing, and that the conjugate is effective onLNCaP tumors (mice used for MTD 2 weeks after implantation of LNCaPcells before treatment was initiated). All 4 mice in the saline controlgroup had large tumors, whereas no mice in the two treated groups hadvisible tumors after 18 days of treatment. (See FIG. 9B)

Example 10

Efficacy study compared to control group and competition group. Animalswere treated with (a) the conjugate SK71 administered in 5 doses onalternate days (M, W, F, M, W) at 1 mol·kg, and compared to (b) vehicletreated animals (FIG. 10B), and to (c) animals treated with theconjugate in conjunction with PMPA. Treatment at 1 mol/kg shows asuccessive decrease in tumor size (starting tumor size approximately 250mm3) during the course of treatment. At the lower dose of 1 μmol/kgshown in FIG. 10A, tumor volumes rebounded at the cessation of dosing.At the higher dose of at least 2 μmol/kg, complete disappearance of thetumor was observed during the testing period. The competitionexperiments (See FIG. 10C) indicate that the successful treatment of theimplanted tumor is related to selective or specific targeting of PSMAmediated delivery.

Example 11

Efficacy Study (1 micromole/kg every other day for 10 days; i.e. 5doses). The data (See FIG. 11) indicate that tumors in treated animalsdecreased in size during the duration of treatment.

Example 12

Evaluation of a PSMA-Targeted Therapeutic Agent in Vitro. Analysis ofSK71 (FIG. 12A), SK77 (FIG. 12B), SK37 (FIG. 12C), and SK45 (FIG. 12D),toxicity to LNCaP cells in culture. LNCaP cells were pulsed for 2 h withincreasing concentrations of SK71 or SK77 in the presence (▴) or absence(▪) of 100-fold molar excess PMPA. After 2× washes, cells were incubatedan additional 66 h in fresh medium at 37° C. Cell viability was thenanalyzed using the [3H]-thymidine incorporation assay, as describedherein. Data were obtained in a single experiment and error barsrepresent s.d. (n=3 wells per concentration).

Example 13

Potency In Vivo. Effect of SK71 on the growth of subcutaneous tumors(FIGS. 13A and 13C, and on the weights of the treated mice (FIGS. 13Band 13D). LNCaP cells in HC Matrigel were implanted subcutaneously intoshoulders of nu/nu male mice. Once tumors reached either 100 mm3 (13A,13B) or 330 mm3 (13C, 13D) in volume, animals were treated with SK71[1.5 μmol/kg (a, b) or 2.0 μmol/kg (c, d)]. Treated mice (▪), untreatedmice (●), treated mice pre-injected with 100-fold (13A, 13B) or 30-fold(13C, 13D) molar excess of PMPA (▴ and ▾, respectively). Data wereobtained in a single experiment and error bars represent s.d. [n=4 (13A,13B) or 3 (13C, 13D)]. FIG. 10. SK71 potency in vivo.

Potency In Vivo. Effect of SK77 on the growth of subcutaneous tumors(FIG. 14A), and on the weights of the treated mice (FIG. 14B). LNCaPcells in HC Matrigel were implanted subcutaneously into shoulders ofnu/nu male mice. Once tumors reached 100 mm3 in volume, animals weretreated with SK77 (2 μmol/kg). Untreated mice (▪), treated mice (▾).Data were obtained in a single experiment and error bars represent s.d.(n=4 mice/group).

What is claimed is:
 1. A compound having the formula B-L-IA, wherein Bis a ligand having the structure

wherein R is selected from the group consisting of —C(O)—, —CH₂C(O)—,and —S— and R is the point of attachment to L, L is a linker selectedfrom the group consisting of ethylene-diamine, ethylene-diamine-glutamicacid, aminocaproyl-phenylalanine-phenylalanine-diaminopropionicacid-aspartic acid-cysteine, and a peptide of two or more amino acids,and IA is an optical imaging agent selected from the group consisting offluorescein, OREGON GREEN fluorescent agents, ALEXAFLUOR fluorescentagents, fluorescein, BODIPY fluorescent agents, rhodamine fluorescentagents, DYLIGHT fluorescent agents, IRDye 800CW, TEXAS RED, andphycoerythrin.
 2. The compound of claim 1, wherein L has a length of atleast 7 atoms.
 3. The compound of claim 1, wherein L has a length of atleast 10 Angstrom.
 4. The compound of claim 1, wherein the OREGON GREENfluorescent agent selected from the group consisting of OREGON GREEN 488and OREGON GREEN
 514. 5. The compound of claim 1, wherein the ALEXAFLUORfluorescent agent selected from the group consisting of ALEXAFLUOR 488and ALEXAFLUOR
 647. 6. The compound of claim 1, wherein the BODIPYfluorescent age selected from the group consisting of BODIPY F1 andBODIPY
 505. 7. The compound of claim 1, wherein the rhodaminefluorescent agent is tetramethylrhodamine.
 8. The compound of claim 1,wherein the DYLIGHT fluorescent agent is DYLIGHT
 680. 9. The compound ofclaim 1, wherein the compound is capable of or adapted to fluoresceafter distribution thereof in tissue.
 10. The compound of claim 9,wherein the tissue is a tumor or a lymph node.
 11. The compound of claim1 wherein the compound is selective for targeting to a tumor cell.
 12. Acomposition comprising the compound of claim 1 and a pharmaceuticallyacceptable carrier, excipient or diluent.